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It is generally thought that venlafaxine raises blood pressure at higher doses; however, some studies have found no effect or a decrease in blood pressure. The aim of this study was to evaluate the cardiovascular (CV) effects of 3 weeks of dosing with venlafaxine, pregabalin and placebo on young healthy adults. Fifty-four participants, of mean age 23.1 years (sd 4.68), 29 male, were randomised into three parallel groups. Each group received one of the three drugs, dosed incrementally over a 3-week period to reach daily doses of 150 mg/day venlafaxine and 200 mg/day pregabalin. Blood pressure sphygmomanometer measurements, heart rate measurements, and orthostatic challenges recorded continuously beat-to-beat were performed weekly over this period and 5 days after treatment cessation. Results showed resting systolic blood pressure (SBP) and resting and standing diastolic blood pressure (DBP) and heart rate (HR) were significantly raised by venlafaxine compared with the pregabalin and placebo groups. SBP drop on standing was larger, the resulting overshoot was smaller, and recovery was slower on venlafaxine. HR recovery was significantly impaired by venlafaxine. CV changes were observed after only 1 week of dosing at 112.5 mg/day. These effects of venlafaxine are likely to be due to its action of noradrenergic reuptake inhibition.

Rationale: The increasing demand to develop more efficient compounds to treat cognitive impairments in schizophrenia has led to the development of experimental model systems. One such model system combines the study of surrogate populations expressing high levels of schizotypy with oculomotor biomarkers., Objectives: We aimed (1) to replicate oculomotor deficits in a psychometric schizotypy sample and (2) to investigate whether the expected deficits can be remedied by compounds shown to ameliorate impairments in schizophrenia., Methods: In this randomized double-blind, placebo-controlled study 233 healthy participants performed prosaccade (PS), antisaccade (AS) and smooth pursuit eye movement (SPEM) tasks after being randomly assigned to one of four drug groups (nicotine, risperidone, amisulpride, placebo). Participants were classified into medium- and high-schizotypy groups based on their scores on the Schizotypal Personality Questionnaire (SPQ, Raine (Schizophr Bull 17:555-564, 1991))., Results: AS error rate showed a main effect of Drug (p < 0.01), with nicotine improving performance, and a Drug by Schizotypy interaction (p = 0.04), indicating higher error rates in medium schizotypes (p = 0.01) but not high schizotypes under risperidone compared to placebo. High schizotypes had higher error rates than medium schizotypes under placebo (p = 0.03). There was a main effect of Drug for saccadic peak velocity and SPEM velocity gain (both p ≤ 0.01) indicating impaired performance with risperidone., Conclusions: We replicate the observation of AS impairments in high schizotypy under placebo and show that nicotine enhances performance irrespective of group status. Caution should be exerted in applying this model as no beneficial effects of antipsychotics were seen in high schizotypes.

Benzodiazepines are prescribed widely but their utility is limited by unwanted side effects, including abuse potential. The mechanisms underlying the abuse-related effects of benzodiazepines are not well understood, although α1 subunit-containing GABAA receptors have been proposed to have a critical role. Here, we examine the reinforcing effects of several compounds that vary with respect to intrinsic efficacy at α2, α3, and α5 subunit-containing GABAA receptors but lack efficacy at α1 subunit-containing GABAA receptors ('α1-sparing compounds'): MRK-623 (functional selectivity for α2/α3 subunit-containing receptors), TPA023B (functional selectivity for α2/α3/α5 subunit-containing receptors), and TP003 (functional selectivity for α3 subunit-containing receptors). The reinforcing effects of the α1-sparing compounds were compared with those of the non-selective benzodiazepine receptor partial agonist MRK-696, and non-selective benzodiazepine receptor full agonists, midazolam and lorazepam, in rhesus monkeys trained to self-administer midazolam or cocaine, under a progressive-ratio schedule of intravenous (i.v.) drug injection. The α1-sparing compounds were self-administered significantly above vehicle levels in monkeys maintained under a midazolam baseline, but not under a cocaine baseline over the dose ranges tested. Importantly, TP003 had significant reinforcing effects, albeit at lower levels of self-administration than non-selective benzodiazepine receptor agonists. Together, these results suggest that α1 subunit-containing GABAA receptors may have a role in the reinforcing effects of benzodiazepine-type compounds in monkeys with a history of stimulant self-administration, whereas α3 subunit-containing GABAA receptors may be important mediators of the reinforcing effects of benzodiazepine-type compounds in animals with a history of sedative-anxiolytic/benzodiazepine self-administration.

Background: Synergistic effects of NK₁ receptor antagonism combined with serotonin reuptake inhibition have been reported in preclinical models. GSK424887 is a selective competitive antagonist of the human NK₁ receptor and inhibitor of the serotonin transporter. However, its actions in human models of depression have not been assessed., Methods: This study explored the effects of acute administration of GSK424887 compared to placebo in healthy male volunteers. The selective serotonin reuptake inhibitor (SSRI) citalopram was used as a positive control. A battery of emotional processing tasks was given at the peak time of drug effect., Results: GSK424887 enhanced attentional vigilance in the dot-probe task to both positive and negative stimuli. By contrast, citalopram enhanced perception of angry, sad and happy facial expressions and increased positive bias in the facial expression recognition task. Neither drug significantly affected emotion potentiated startle responses or emotional memory., Conclusions: These results suggest that acute administration of GSK424887 modulated some aspects of emotional processing but these effects were not similar to those seen previously with antidepressant agents. This was the first use of the battery of emotional processing tasks in a Phase 1 study. Repeated administration of the test and active control drugs may be needed to reliably characterise their effects.

Previous studies have shown that subjective and objective symptoms of anxiety induced by 7.5% CO(2) inhalation can be attenuated by anxiolytics such as lorazepam and, to a lesser extent, paroxetine. Venlafaxine and pregabalin, two other licensed treatments for Generalised Anxiety Disorder, were used to further investigate the 7.5% and 35% CO(2) models of anxiety in healthy volunteers. Fifty-four participants were randomised to receive either placebo, venlafaxine or pregabalin. Study treatments were dosed incrementally over a three week period, to reach daily doses of 150 mg venlafaxine and 200mg pregabalin by the CO(2) challenge test day. Participants inhaled air 7.5% CO(2) for 20 minutes (single-blind presentation), and a non-blinded single vital capacity of 35% CO(2). Subjective ratings were recorded before and after each inhalation. Both 7.5% and 35% CO(2) inhalations produced the expected effects of increased ratings of symptoms of panic and anxiety, with increased blood pressure and heart rate. No significant treatment effects were found, although there were trends towards a reduction in feeling tense and nervous by both drugs compared with placebo during the 7.5% CO(2) challenge, and a reduction in alertness generally in the venlafaxine group compared with the pregabalin group. In contrast with the clear anxiolytic effects of benzodiazepines reported in several previous CO(2) studies, these findings suggest that the anxiogenic effects of CO(2) challenges are not significantly influenced by these serotonergic and GABAergic anxiolytics. This may be due to a lack of sensitivity of the CO(2) challenges in healthy volunteers to these drug types.

Symptoms of anxiety induced by 7.5% CO₂ inhalation can be attenuated by acute administration of GABA(A) receptor anxiolytics such as lorazepam and alprazolam. This study investigated if these effects are dose-related, by comparing a 0.5 mg dose (considered non-clinically effective) and a 2 mg dose of lorazepam (clinically effective) on 7.5% CO₂ inhalation. Eighteen healthy males (mean age 20.6 years, SD 1.29), judged physically and mentally fit, attended three visits, each one week apart, to take each treatment in a randomised double-blind crossover design. Drugs were given 60 min prior to 20 min air inhalation, followed by 20 min 7.5% CO₂ inhalation. The order of gas presentation was single blind. Subjective ratings using visual analogue scales (VAS) and questionnaires were recorded before and after each inhalation. Blood pressure (BP), heart rate (HR), respiration rate (RR) and expired CO₂ were recorded during each inhalation. Inhalation of 7.5% CO₂ significantly raised BP, HR, RR and expired CO₂. Ratings of feeling like leaving the room were significantly lower on 2 mg compared with 0.5 mg and placebo, and dose-dependent trends were seen in scores for VAS fearful, anxious, stressed, tense, and worried. Results may be indicative of dose-dependent effects of lorazepam in a CO₂ model of anxiety., (Copyright © 2012 John Wiley & Sons, Ltd.)

A number of compounds aimed at improving cognition in schizophrenia have failed to demonstrate efficacy in Phase 2 clinical trials. Translational studies using biomarkers in surrogate populations, such as schizotypy, could be used to assess the efficacy of novel compounds. In this study, we aimed to validate the sensitivity and inter-site reliability of cognitive biomarkers (working memory (N-back), spatial working memory (SWM) and verbal fluency (VF) tasks) to detect the schizotypy phenotype and its reversal by psychotropic drugs. Healthy volunteers scoring high or average on a schizotypal personality measure (122 in each group) were randomized to receive a single dose of risperidone, amisulpride, nicotine or placebo in a double-blind, between-subject design. We found evidence for a poorer performance on N-back and VF tasks in the high schizotypy group, replicating previous research. This effect was counteracted by amisulpride on N-back: it improved working memory in high schizotypy group but impaired the controls. A similar pattern was seen in SWM and VF. We interpret this finding in the light of the dopamine enhancing action of amisulpride when given in low doses. In contrast, risperidone impaired both groups and nicotine had a beneficial effect for the low baseline performers only. These effects were consistent across sites. These data demonstrates the utility of biomarkers in detecting the effect of schizotypy and its reversal by drugs that enhance dopamine and cholinergic function. Studies using similar design could help the early assessment of potential of compounds designed to improve cognition in schizophrenia., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Agomelatine is a new antidepressant with a novel profile of pharmacological action. The clinical efficacy of agomelatine has been established in major depression, but its actions on emotional bias are unknown. Consequently, the current experimental study assessed the effect of agomelatine on emotional processing in healthy volunteers using an Emotional Test Battery shown to be sensitive to serotonin and noradrenaline reuptake inhibitors. Volunteers were randomized to receive placebo, 25 mg or 50 mg of agomelatine over a 7-day period in a double-blind parallel groups design. Emotional processing (n = 48) was assessed on the morning of day 8 using the Emotional Test Battery which included facial expression recognition, emotional memory, attentional visual probe and emotion-potentiated startle. Mood and subjective state were monitored before and during treatment. Agomelatine (25 mg) decreased subjective ratings of sadness, reduced recognition of sad facial expressions, improved positive affective memory and reduced the emotion-potentiated startle response. The results show that agomelatine has more selective effects on the processing of social facial cues than conventional antidepressants, which could contribute to less blunting of emotional experience. The study highlights the potential value of volunteer models in drug development for screening and profiling of novel antidepressants.

In the pharmaceutical industry deciding whether to progress a compound to the next stage of development or choosing between compounds in a development portfolio is laden with risk. This is particularly true of compounds developed to treat CNS disorders. The use of pre-clinical models in CNS drug development is well established but these models often lack predictive validity and many compounds fail when they reach the target patient group. Bridging the gap between pre-clinical CNS models and patient studies, P1vital's objective is to develop human volunteer models that will enable rapid, accurate and reliable decision making about which compounds to progress into patient trials. The research strategy of P1vital and its academic research network is to focus on science that progresses the development of clinical efficacy models. As part of this strategy P1vital established a CNS Experimental Medicine Consortium with members from both academic research and the pharmaceutical industry. This consortium is unique in that experimental medicine models initially developed through academic research are selected for further validation in a process that is managed by the Pharma members of the P1vital CNS Experimental Medicine Consortium steering (PEM) committee. The P1vital consortium is very much a work in progress. However, since its inception in 2007 the consortium has successfully delivered results from five clinical studies in four therapeutic areas namely, anxiety, cognitive disorders, schizophrenia and depression., (2011. Published by Elsevier Inc.)

Rationale: Experimental evidence suggests that the differential behavioral effects of benzodiazepines depend on their relative actions at γ-aminobutyric acid type A (GABA(A)) receptors that contain either an α1, α2, α3, or α5 subunit., Objectives: The present study was aimed at understanding the role of α3 subunit-containing GABA(A) (α3GABA(A)) receptors by examining the behavioral pharmacology of TP003 (4,2'-difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-a]pyridine-3-yl]biphenyl-2-carbonitrile), which shows functional selectivity for α3GABA(A) receptors., Methods: First, a conflict procedure was used to assess the anxiolytic-like effects of TP003 and a representative clinically available benzodiazepine. TP003 was also administered before daily periods of sucrose pellet availability to evaluate potential hyperphagic effects. In separate experiments, observable behavioral effects were used to assess the motor and sedative effects of TP003., Results: Administration of TP003 produced robust anti-conflict effects without the rate-decreasing effects that were observed with the representative benzodiazepine. Unlike the reported effects of benzodiazepines, TP003 did not enhance palatable food consumption. However, increases in observable sleep-associated posture were induced by TP003, as were decreases in some species-typical behaviors (vocalization, locomotion, and environment-directed behaviors). When evaluated for its ability to induce a procumbent posture, TP003 failed to produce an effect., Conclusions: Based on conflict and observation tests in monkeys, our results suggest that TP003 may have anxiolytic properties but lack ataxic, hyperphagic, and pronounced sedative effects characteristic of classical benzodiazepines. TP003 did induce myorelaxant-like effects and had relatively mild sedative effects. Collectively, these results suggest that α3GABA(A) receptors play an important role in the anxiolytic-like and motor effects of benzodiazepine-type drugs.

In the accompanying paper we describe how MRK-409 unexpectedly produced sedation in man at relatively low levels of GABA(A) receptor occupancy (∼10%). Since it was not clear whether this sedation was mediated via the α2/α3 or α1 GABA(A) subtype(s), we characterized the properties of TPA023B, a high-affinity imidazotriazine which, like MRK-409, has partial agonist efficacy at the α2 and α3 subtype but is an antagonist at the α1 subtype, at which MRK-409 has weak partial agonism. TPA023B gave dose- and time-dependent occupancy of rat brain GABA(A) receptors as measured using an in vivo [(3)H]flumazenil binding assay, with 50% occupancy corresponding to a respective dose and plasma drug concentration of 0.09 mg/kg and 19 ng/mL, the latter of which was similar to that observed in mice (25 ng/mL) and comparable to values obtained in baboon and man using [(11)C]flumazenil PET (10 and 5.8 ng/mL, respectively). TPA023B was anxiolytic in rodent and primate (squirrel monkey) models of anxiety (elevated plus maze, fear-potentiated startle, conditioned suppression of drinking, conditioned emotional response) yet had no significant effects in rodent or primate assays of ataxia and/or myorelaxation (rotarod, chain-pulling, lever pressing), up to doses (10 mg/kg) corresponding to occupancy of greater than 99%. In man, TPA023B was well tolerated at a dose (1.5 mg) that produced occupancy of >50%, suggesting that the sedation previously seen with MRK-409 is due to the partial agonist efficacy of that compound at the α1 subtype, and highlighting the importance of antagonist efficacy at this particular GABA(A) receptor population for avoiding sedation in man.

MRK-409 binds to α1-, α2-, α3- and α5-containing human recombinant GABA(A) receptors with comparable high affinity (0.21-0.40 nM). However, MRK-409 has greater agonist efficacy at the α3 compared with α1 subtypes (respective efficacies relative to the full agonist chlordiazepoxide of 0.45 and 0.18). This compound readily penetrates the brain in rats and occupies the benzodiazepine site of GABA(A) receptors, measured using an in vivo [(3)H]flumazenil binding assay, with an Occ(50) of 2.2 mg/kg p.o. and a corresponding plasma EC(50) of 115 ng/mL. Behaviourally, the α3-preferring agonist efficacy profile of MRK-409 produced anxiolytic-like activity in rodent and primate unconditioned and conditioned models of anxiety with minimum effective doses corresponding to occupancies, depending on the particular model, ranging from ∼35% to 65% yet there were minimal overt signs of sedation at occupancies greater than 90%. In humans, however, safety and tolerability studies showed that there was pronounced sedation at a dose of 2 mg, resulting in a maximal tolerated dose of 1 mg. This 2 mg dose corresponded to a C(max) plasma concentration of 28 ng/mL, which, based on the rodent plasma EC(50) for occupancy of 115 ng/mL, suggested that sedation in humans occurs at low levels of occupancy. This was confirmed in human positron emission tomography studies, in which [(11)C]flumazenil uptake following a single dose of 1 mg MRK-409 was comparable to that of placebo, indicating that occupancy of GABA(A) receptor benzodiazepine binding sites by MRK-409 was below the limits of detection (i.e. <10%). Taken together, these data show that MRK-409 causes sedation in humans at a dose (2 mg) corresponding to levels of occupancy considerably less than those predicted from rodent models to be required for anxiolytic efficacy (∼35-65%). Thus, the preclinical non-sedating anxiolytic profile of MRK-409 did not translate into humans and further development of this compound was halted.

GABA(A) receptor α5-selective inverse agonists enhance cognitive performance in pre-clinical species. However, a key aspect of the clinical development of such compounds is the demonstration that in man such compounds are devoid of the anxiogenic-like activity associated with non-selective inverse agonists such as FG 7142. The triazolophthalazine α5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is an α5-selective inverse agonist which enhances cognitive performance in rodents and encouragingly in human Phase I Safety and Tolerability studies it was devoid of the anxiogenic-like activity associated with FG 7142. However, in order to appropriately interpret this latter observation, it was considered important to demonstrate that the absence of anxiogenic-like activity occurs at significant levels of receptor occupancy. Consequently, the occupancy of human brain GABA(A) receptors was measured using [¹¹C]flumazenil positron emission tomography in three healthy normal young male volunteers following a single oral dose of 2 mg α5IA. One hour after dosing, mean occupancy levels were 53% and this fell to 16% by 8 h post-dose, with the plasma α5IA concentration corresponding to 50% occupancy being 10 ng/mL. These data clearly show that an α5-selective inverse agonist is not associated with anxiogenic-like side effects at doses that give ~50% occupancy., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Previous reports suggest that gamma-aminobutyric acid type A (GABA(A)) receptors containing alpha1 subunits may play a pivotal role in mediating the discriminative stimulus effects of benzodiazepines (BZs). L-838,417 (7-tert-Butyl-3-(2,5-difluoro-phenyl)-6-(2-methyl-2H-[1,2,4]triazol-3-ylmethoxy)-[1,2,4]triazolo[4,3-b]pyridazine) is a GABA(A) receptor modulator with intrinsic efficacy in vitro at alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors, and little demonstrable intrinsic efficacy in vitro at alpha1 subunit-containing GABA(A) receptors. The present study evaluated the discriminative stimulus effects of L-838,417 in order to determine the extent to which the alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors contribute to the interoceptive effects of BZ-type drugs. Squirrel monkeys (Saimiri sciureus) were trained to discriminate L-838,417 (0.3 mg/kg, i.v.) from vehicle under a 5-response fixed-ratio schedule of food reinforcement. Under test conditions, L-838,417 administration resulted in dose-dependent increases in drug-lever responding that were antagonized by the BZ-site antagonist, flumazenil. Administration of non-selective BZs, compounds with 10-fold greater affinity for alpha1 subunit-containing GABA(A) receptors compared to alpha2, alpha3, and alpha5 subunit-containing GABA(A) receptors, barbiturates and ethanol (which modulate the GABA(A) receptor via a non-BZ site), all resulted in a majority of responses on the L-838,417-paired lever (65-100% drug-lever responding). betaCCT, an antagonist that binds with 20-fold greater affinity for alpha1 subunit-containing GABA(A) receptors relative to alpha2, alpha3, and alpha5-containing GABA(A) receptors, had no significant effect on the discriminative stimulus effects of L-838,417 or the L-838,417-like effects of diazepam or zolpidem. These data suggest that efficacy at alpha2, alpha3, and/or alpha5 subunit-containing GABA(A) receptors likely are sufficient for engendering BZ-like discriminative stimulus effects., (2009 Elsevier Ltd. All rights reserved.)

Abuse-liability-related effects of subtype-selective GABA(A) modulators were explored relative to the prototypic benzodiazepine lorazepam. 7-Cyclobutyl-6-(2-methyl-2H-1,2,4-triazol-3-ylmethoxy)-3-phenyl-1,2,4-triazolo[4,3-b]pyridazine (TPA123) has weak partial agonist efficacy at alpha(1)-, alpha(2)-, alpha(3)-, and alpha(5)-containing GABA(A) receptors, whereas 7-(1,1-dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) has weaker partial agonist efficacy at alpha(2) and alpha(3) and none at alpha(1) and alpha(5) subtypes. For both compounds, preclinical data suggested efficacy as nonsedating anxiolytics. Self-injection of TPA123 (0.0032-0.1 mg/kg) and TPA023 (0.0032-0.32 mg/kg) was compared with lorazepam (0.01-0.32 mg/kg) in baboons. TPA123 and lorazepam maintained self-injection higher than vehicle at two or more doses in each baboon; peak rate of self-injection of lorazepam was higher than TPA123. Self-injected lorazepam and TPA123 also increased rates of concurrently occurring food-maintained behavior. After the availability of self-administered TPA123 doses ended, an effect consistent with a mild benzodiazepine-like withdrawal syndrome occurred. In contrast with lorazepam and TPA123, TPA023 did not maintain self-administration. Positron emission tomography studies showed that TPA023 produced a dose-dependent inhibition in the binding of [(11)C]flumazenil to the benzodiazepine binding site in the baboon, which was essentially complete (i.e., 100% occupancy) at the highest TPA023 dose (0.32 mg/kg). In a physical dependence study, TPA023 (32 mg/kg/24 h) was delivered as a continuous intragastric drip. Neither flumazenil at 14 days nor stopping TPA023 after 30 to 31 days resulted in the marked withdrawal syndrome characteristic of benzodiazepines in baboons. In the context of other data, elimination of efficacy at the alpha(1) subtype of the GABA/benzodiazepine receptor is not sufficient to eliminate abuse liability but may do so when coupled with reduced alpha(2/3) subtype efficacy.

Given the suggestion that many potential anti-obesity drugs may enhance within-meal satiation, few studies have directly measured the effects of any drug on the microstructure of human eating behaviour. The effects of 7 days dosing with sibutramine 10 mg and 15 mg a day on appetite and energy balance were determined in 30 obese women (BMI 34.6 +/- 3.3 kg/m2, age 46.0 +/- 12.9 years) using a Universal Eating Monitor (UEM) and indirect calorimetry, in a double-blind, placebo-controlled crossover study. At day 7, sibutramine 10 mg and 15 mg reduced food intake by 16.6% and 22.3%, respectively (p < 0.001), compared with placebo. Sibutramine reduced eating rate compared with placebo rather than meal length (10 mg p < 0.05; 15 mg p < 0.001). In addition, sibutramine 10 mg significantly reduced hunger later in the meal (p < 0.05) and sibutramine 15 mg increased fullness early in the meal (p < 0.01), both of which are consistent with enhanced within-meal satiation. Sibutramine had little effect on resting metabolic rate, although 15 mg did significantly reduce respiratory quotient at several time points during the test day. These results provide novel evidence that decreased consumption of a test meal induced by sibutramine is primarily because of reduced eating rate, enhancing the deceleration in cumulative food intake within a meal associated with the development of satiety. Changes in within-meal appetite ratings appear particularly sensitive to drug-induced enhancement of satiation, and may provide key indices for assessing the therapeutic potential of novel anti-obesity drugs.

3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)-pyrazolo[1,5-d][1,2,4]triazine (MRK-016) is a pyrazolotriazine with an affinity of between 0.8 and 1.5 nM for the benzodiazepine binding site of native rat brain and recombinant human alpha1-, alpha2-, alpha3-, and alpha5-containing GABA(A) receptors. It has inverse agonist efficacy selective for the alpha5 subtype, and this alpha5 inverse agonism is greater than that of the prototypic alpha5-selective compound 3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-hdyl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine (alpha5IA). Consistent with its greater alpha5 inverse agonism, MRK-016 increased long-term potentiation in mouse hippocampal slices to a greater extent than alpha5IA. MRK-016 gave good receptor occupancy after oral dosing in rats, with the dose required to produce 50% occupancy being 0.39 mg/kg and a corresponding rat plasma EC(50) value of 15 ng/ml that was similar to the rhesus monkey plasma EC(50) value of 21 ng/ml obtained using [(11)C]flumazenil positron emission tomography. In normal rats, MRK-016 enhanced cognitive performance in the delayed matching-to-position version of the Morris water maze but was not anxiogenic, and in mice it was not proconvulsant and did not produce kindling. MRK-016 had a short half-life in rat, dog, and rhesus monkey (0.3-0.5 h) but had a much lower rate of turnover in human compared with rat, dog, or rhesus monkey hepatocytes. Accordingly, in human, MRK-016 had a longer half-life than in preclinical species ( approximately 3.5 h). Although it was well tolerated in young males, with a maximal tolerated single dose of 5 mg corresponding to an estimated occupancy in the region of 75%, MRK-016 was poorly tolerated in elderly subjects, even at a dose of 0.5 mg, which, along with its variable human pharmacokinetics, precluded its further development.

Background and Purpose: alpha5IA (3-(5-methylisoxazol-3-yl)-6-[(1-methyl-1,2,3-triazol-4-yl)methyloxy]-1,2,4-triazolo[3,4-a]phthalazine) is a triazolophthalazine with subnanomolar affinity for alpha1-, alpha2-, alpha3- and alpha5-containing GABA(A) receptors. Here we have evaluated the relationship between plasma alpha5IA concentrations and benzodiazepine binding site occupancy in rodents and primates (rhesus monkey)., Experimental Approach: In awake rats, occupancy was measured at various times after oral dosing with alpha5IA (0.03-30 mgxkg(-1)) using an in vivo {[(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)} binding assay. In anaesthetized rhesus monkeys, occupancy was measured using {[(123)I]iomazenil (ethyl 5,6-dihydro-7-iodo-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylic acid ethyl ester)} gamma-scintigraphy and a bolus/infusion paradigm. In both rat and rhesus monkey, the plasma drug concentration corresponding to 50% occupancy (EC(50)) was calculated., Key Results: In rats, alpha5IA occupancy was dose- and time-dependent with maximum occupancy occurring within the first 2 h. However, rat plasma EC(50) was time-independent, ranging from 42 to 67 ngxmL(-1) over a 24 h time course with the average being 52 ngxmL(-1) (i.e. occupancy decreased as plasma drug concentrations fell). In rhesus monkeys, the EC(50) for alpha5IA displacing steady-state [(123)I]iomazenil binding was 57 ngxmL(-1)., Conclusions and Implications: Rat plasma EC(50) values did not vary as a function of time indicating that alpha5IA dissociates readily for the GABA(A) receptor in vivo. These data also suggest that despite the different assays used (terminal assays of [(3)H]flumazenil in vivo binding in rats and [(123)I]iomazenil gamma-scintigraphy in anaesthetized rhesus monkeys), these techniques produced similar plasma alpha5IA EC(50) values (52 and 57 ngxmL(-1) respectively) and that the plasma-occupancy relationship for alpha5IA translates across these two species.

Age-related decline in allocentric (viewer-independent) spatial memory is seen across species. We employed a virtual reality analogue of the Morris Water Maze to study the effect of healthy ageing on neural activity during allocentric spatial memory using functional magnetic resonance imaging. Voxel-based morphometry was used to ascertain hippocampal volumetric integrity. A widespread neural network comprising frontal, parietal, occipital, thalamic, and cerebellar regions was activated in young and older adults, but only young adults significantly activated bilateral hippocampus and left parahippocampus, as well as right frontal pole and dorso-lateral prefrontal cortex (DLPFC) during encoding and right DLPC during retrieval. Hippocampal grey matter volume was unchanged in older adults; however, prefrontal and parahippocampal functional attenuation was accompanied by volumetric reduction. We conclude that the decline in allocentric spatial memory with age is associated with attenuated hippocampal function, as well as compromised function and structure of prefrontal and parahippocampal regions.

alpha2 subunit-containing GABA(A) receptors are involved in incentive learning associated with cocaine, and in cocaine addiction. Deletion of alpha2-containing receptors abolishes cocaine-induced behavioural sensitisation (BS), while selective activation of alpha2 receptors, achieved using Ro 15-4513's agonist properties in alpha2(H101R) mice, induced BS. Here, we investigate further the mechanisms underlying Ro 15-4513-induced behavioural sensitisation in alpha2(H101R) mice. alpha2(H101R) mice sensitised to Ro 15-4513 (10 mg/kg) showed an enhanced stimulant response to cocaine (10 mg/kg). In contrast, cocaine (10 mg/kg)-sensitised alpha2(H101R) mice did not show enhanced sensitivity to the stimulant effects of Ro 15-4513 (1, 3 and 10 mg/kg), suggesting that the neural adaptations underlying Ro 15-4513 induced BS are related to, but not identical with those associated with cocaine-induced plasticity. Secondly, we investigated whether alpha2-containing receptors are involved in mediating the ability of BZs to facilitate cocaine-induced activity. The non-selective (i.e., alpha1, alpha2, alpha3 and alpha5 subtype) benzodiazepine GABA(A) receptor agonist midazolam (10 and 30 mg/kg) potentiated cocaine (10 mg/kg) hyperactivity in wildtype mice, but not in alpha2(H101R) mice, in which alpha2-containing receptors are insensitive to benzodiazepines. To determine where alpha2 receptors are localised we compared BZ-insensitive sites between wildtype (alpha4 and alpha6) and alpha2(H101R) (alpha2, alpha4 and alpha6) mice, using quantitative autoradiography to estimate [(3)H]Ro 15-4513 binding in the presence of 10 muM diazepam. alpha2 receptors were found in projection areas of the mesolimbic dopamine pathway including accumbens, central amygdala, and basolateral amygdala as well as CA1 and CA3 areas of the hippocampus. The involvement of the alpha2-containing receptor in mediating BZ's potentiating effect on cocaine hyperactivity suggests that the locomotor stimulant effects of BZs and psychostimulants may be mediated by a common neural system, but the lack of cross sensitisation to Ro 15-4513 in cocaine-sensitised alpha2(H101R) mice, suggests that this form of BS may occur downstream of plastic events underlying cocaine sensitisation.

Alcohol produces many subjective and objective effects in man including pleasure, sedation, anxiolysis, plus impaired eye movements and memory. In human volunteers we have used a newly available GABA-A/benzodiazepine receptor inverse agonist that is selective for the alpha5 subtype (a5IA) to evaluate the role of this subtype in mediating these effects of alcohol on the brain. After pre-treatment with a5IA, we found almost complete blockade of the marked impairment caused by alcohol (mean breath concentration 150mg/100ml) of word list learning and partial but non-significant reversal of subjective sedation without effects on other measures such as intoxication, liking, and slowing of eye movements. This action was not due to alterations in alcohol kinetics and so provides the first proof of concept that selectively decreasing GABA-A receptor function at a specific receptor subtype can offset some actions of alcohol in humans. It also supports growing evidence for a key role of the alpha5 subtype in memory. Inverse agonists at other GABA-A receptor subtypes may prove able to reverse other actions of alcohol, and so offer a new approach to understanding the actions of alcohol in the human brain and in the treatment of alcohol related disorders in humans.

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.

Rationale: Compounds selective for the GABAA receptors containing an alpha5 subunit have been reported to enhance performance in the hippocampally mediated delayed-matching-to-position version of the Morris water maze, in which reduction in the time required to find a hidden platform relative to an initial trial is used as an index of learning and memory., Objective: In the present study, we have used one such compound, alpha5IA-II, to examine whether these effects occur during the encoding, consolidation or recall phases of this paradigm., Methods: alpha5IA-II was administered in the absence or presence of the benzodiazepine site antagonist flumazenil, so as to limit its action to periods associated with encoding, consolidation and recall. Drug doses and timings of administrations were defined using occupancy data derived from an in vivo [3H]flumazenil binding assay. Similar experiments were carried out to study the memory-disruptive properties of chlordiazepoxide (CDP)., Results: The trial 1 to trial 2 difference was increased when alpha5IA-II was given before either trial 1 or trial 2, indicating an effect on the encoding and recall phases, respectively, of learning and memory. Conversely, alpha5IA-II had no effect on performance when given immediately after trial 1, suggesting that it had no effect on the consolidation phase. In contrast to the facilitation of performance produced by the alpha5-selective inverse agonist alpha5IA-II given during the encoding and recall but not the consolidation phase, the non-selective agonist CDP impaired performance when given during the encoding and recall phases, whilst having no effect on the consolidation phase., Conclusions: These data further highlight the cognition-enhancing properties of GABAA alpha5-selective inverse agonists and define the functional specificity of these effects in terms of encoding and recall processes in the Morris water maze.

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.

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.

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.

Mice with point-mutated alpha2 GABAA receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in unconditioned models of anxiety. We investigated the role of the alpha2 GABAA subtype in a model of conditioned anxiety. alpha2(H101R) and wildtype mice were trained in a conditioned emotional response (CER) task, in which lever-pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a conditioned stimulus (CS+) that predicted footshock. The ability of diazepam, ethanol and pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. Diazepam (0, 0.5, 1, 2, 4 and 8 mg/kg) induced a dose-dependent anxiolytic-like effect in wildtype mice. At high doses, diazepam (2, 4 and 8 mg/kg) was sedative in alpha2(H101R) mice. Analysis of the anxiolytic properties of nonsedative diazepam doses (0.5 and 1 mg/kg), showed that alpha2(H101R) mice were resistant to the anxiolytic effects of diazepam. Equivalent anxiolytic properties of pentobarbital (20 mg/kg) and ethanol (1 and 2 g/kg) were seen in both genotypes. These findings confirm the critical importance of the alpha2 GABAA subtype in mediating BZ anxiolysis. However, as a compound, L-838417, with agonist properties at alpha2, alpha3 and alpha5-containing receptors, gave rise to anxiolytic-like activity in alpha2(H101R) mice in the CER test, alpha3-containing GABA receptors are also likely to contribute to anxiolysis. Observations that alpha2(H101R) mice were more active, and displayed a greater suppression of lever pressing in response to fear-conditioned stimuli than wildtype mice, suggests that the alpha2(H101R) mutation may not be behaviourally silent.

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.

Alpha5IA is a compound that binds with equivalent subnanomolar affinity to the benzodiazepine (BZ) site of GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has inverse agonist efficacy selective for the alpha5 subtype. As a consequence, the in vitro and in vivo effects of this compound are mediated primarily via GABA(A) receptors containing an alpha5 subunit. In a mouse hippocampal slice model, alpha5IA significantly enhanced the burst-induced long-term potentiation of the excitatory postsynaptic potential in the CA1 region but did not cause an increase in the paroxysmal burst discharges that are characteristic of convulsant and proconvulsant drugs. These in vitro data suggesting that alpha5IA may enhance cognition without being proconvulsant were confirmed in in vivo rodent models. Hence, alpha5IA significantly enhanced performance in a rat hippocampal-dependent test of learning and memory, the delayed-matching-to-position version of the Morris water maze, with a minimum effective oral dose of 0.3 mg/kg, which corresponded to a BZ site occupancy of 25%. However, in mice alpha5IA was not convulsant in its own right nor did it potentiate the effects of pentylenetetrazole acutely or produce kindling upon chronic dosing even at doses producing greater than 90% occupancy. Finally, alpha5IA was not anxiogenic-like in the rat elevated plus maze nor did it impair performance in the mouse rotarod assay. Together, these data suggest that the GABA(A) alpha5-subtype provides a novel target for the development of selective inverse agonists with utility in the treatment of disorders associated with a cognitive deficit.

7-(1,1-Dimethylethyl)-6-(2-ethyl-2H-1,2,4-triazol-3-ylmethoxy)-3-(2-fluorophenyl)-1,2,4-triazolo[4,3-b]pyridazine (TPA023) is a triazolopyridazine that binds with equivalent high (subnanomolar) affinity to the benzodiazepine binding site of recombinant human GABA(A) receptors containing an alpha1, alpha2, alpha3, or alpha5 subunit but has partial agonist efficacy at the alpha2 and alpha3 subtypes and essentially antagonist efficacy at the alpha1 and alpha5 subtypes. In rats, TPA023 gave time- and dose-dependent occupancy after oral dosing, with 50% occupancy corresponding to a dose of 0.42 mg/kg. It has anxiolytic-like activity in unconditioned (elevated plus maze) and conditioned (fear-potentiated startle and conditioned suppression of drinking) rat models of anxiety with minimum effective doses (MED; 1-3 mg/kg) corresponding to 70 to 88% occupancy. However, there was no appreciable sedation in a response sensitivity (chain-pulling) assay at a dose of 30 mg/kg, resulting in 99% occupancy. Similarly, TPA023 was robustly anxiolytic in the squirrel monkey conditioned emotional response assay, with a MED of 0.3 mg/kg, but did not produce any sedation in a lever-pressing test of sedation even at 10 mg/kg. TPA023 produced no impairment in performance in the mouse Rotarod assay, and there was only a mild interaction with ethanol. In addition to anxiolytic-like efficacy, TPA023 had anticonvulsant activity in a mouse pentylenetetrazole seizure model. Finally, TPA023 did not cause precipitated withdrawal in mice treated for 7 days with the nonselective agonist triazolam, nor did N-methyl-beta-carboline-3-carboxamide (FG 7142) precipitate withdrawal in mice treated for 7 days with TPA023. In summary, the novel alpha2/alpha3-selective efficacy profile of TPA023 translates into a nonsedating anxiolytic profile that is distinct from nonselective agonists.

GABA(A) receptors containing alpha5 subunits have been suggested to mediate the rewarding effects of ethanol. We tested this hypothesis in mice with deletion of alpha5 subunits. alpha5 knockout mice did not differ from wildtypes in operant responding for 10% ethanol/10% sucrose, but responded less for 10% sucrose. The benzodiazepine (BZ) site inverse agonist, Ro 15-4513, has higher affinity for GABA(A) receptors containing 5 subunits and dose-dependently (0-27 mg/kg, i.p.) reduced lever pressing for ethanol/sucrose in wildtype mice, but had less effect in knockout mice; lever pressing for sucrose was unaffected. These data suggest that alpha5 subunits are not essential for ethanol reward, but the reduction of operant responding for ethanol by Ro 15-4513 is mediated by alpha5-containing GABA(A) receptors. In measures of ethanol consumption, alpha5 knockout mice did not differ from wildtypes at low ethanol concentrations (2-8%), but consumed less ethanol at higher concentrations; these differences were not attributable to increased behavioural disruption of the knockout by ethanol, since no differences were seen in sensitivity to ethanol's sedative or ataxic effects. Ro 15-4513's ability to reduce ethanol consumption was unaffected, suggesting that this effect is not mediated by the alpha5 subtype. Secondly, we tested the ability of a novel alpha5-efficacy-selective benzodiazepine receptor ligand, alpha5IA-II, that possesses greater inverse agonist activity at alpha5- than at alpha1-, á2- or alpha3-containing GABA(A) receptors, to influence operant responding. alpha5IA-II (0.03-3 mg/kg) dose-dependently decreased lever pressing for 10% ethanol, the minimally effective dose of 1 mg/kg, corresponding to over 90% receptor occupancy, but did not affect lever pressing for 4% sucrose. Although inverse agonists acting at alpha5-containing receptors reduce ethanol self-administration, alpha5 subunits may not be essential to signaling ethanol reward.

There is increasing evidence that compounds with selectivity for gamma-aminobutyric acid(A) (GABA(A)) alpha2- and/or alpha3-subtypes may retain the desirable anxiolytic activity of nonselective benzodiazepines but possess an improved side effect profile. Herein we describe a novel series of GABA(A) alpha2/alpha3 subtype-selective agonists leading to the identification of the development candidate 17, a nonsedating anxiolytic in preclinical animal assays.

The GABA(A) receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that alpha2-rather than alpha3-containing GABA(A) receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an alpha3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2'-difluoro-5'-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an alpha3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders alpha2-containing receptors BZ insensitive (alpha2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of alpha3-containing GABA(A) receptors is sufficient to produce the anxiolytic effects of BZs and that alpha2 potentiation may not be necessary.

Learning and memory are central topics in behavioral neuroscience, and inbred mice strains are widely investigated. However, operant conditioning techniques are not as extensively used in this field as they should be, given the effectiveness of the methodology of the experimental analysis of behavior. In the present study, male C57B1/6 mice, widely used as background for transgenic studies, were trained to lever press on discrete-trial fixed-ratio 5 or fixed-interval (11 s or 31 s) schedules of food reinforcement and then exposed to 15 extinction sessions following vehicle or chlordiazepoxide injections (15 mg/kg i.p., administered either prior to all extinction sessions, or prior to the final 10 extinction sessions). Extinction of operant behavior was facilitated by drug administration following training on either schedule, but this facilitation only occurred once a number of extinction sessions had taken place. The extinction process proceeded more rapidly following fixed-interval training. Resistance to extinction was equally high following training with either schedule type, and was reduced by drug administration in both cases. These phenomena were evident in individual cumulative records and in analyses of group data. Results are interpreted in terms of phenomena of operant extinction identified in Skinner's (1938) Behavior of Organisms, and by behavioral momentum theory. These procedures could be used to extend the contribution of operant conditioning to contemporary behavioral neuroscience.

The mouse rotarod test of motor coordination/sedation is commonly used to predict clinical sedation caused by novel drugs. However, past experience suggests that it lacks the desired degree of sensitivity to be predictive of effects in humans. For example, the benzodiazepine, bretazenil, showed little impairment of mouse rotarod performance, but marked sedation in humans. The aim of the present study was to assess whether the mouse beam walking assay demonstrates: (i) an increased sensitivity over the rotarod and (ii) an increased ability to predict clinically sedative doses of benzodiazepines. The study compared the effects of the full benzodiazepine agonists, diazepam and lorazepam, and the partial agonist, bretazenil, on the mouse rotarod and beam walking assays. Diazepam and lorazepam significantly impaired rotarod performance, although relatively high GABA-A receptor occupancy was required (72% and 93%, respectively), whereas beam walking performance was significantly affected at approximately 30% receptor occupancy. Bretazenil produced significant deficits at 90% and 53% receptor occupancy on the rotarod and beam walking assays, respectively. The results suggest that the mouse beam walking assay is a more sensitive tool for determining benzodiazepine-induced motor coordination deficits than the rotarod. Furthermore, the GABA-A receptor occupancy values at which significant deficits were determined in the beam walking assay are comparable with those observed in clinical positron emission tomography studies using sedative doses of benzodiazepines. These data suggest that the beam walking assay may be able to more accurately predict the clinically sedative doses of novel benzodiazepine-like drugs.

It is well known that modafinil is an effective wake-promoting agent, but there is growing evidence to suggest that modafinil may also enhance some aspects of cognition. In man, modafinil has been shown to enhance vigilance in sleep-deprived and/or narcoleptic subjects and also to improve executive-type functioning (predominantly inhibitory response control processes) across a variety of human patient population groups. Preclinically, a delay-dependent improvement has been reported with modafinil in a mouse T-maze test of working memory. To investigate further the role of modafinil as a potential cognition enhancer, the effects of modafinil on attentional processes were assessed in the rat. The aim of the present study was to evaluate the potential of modafinil to enhance five-choice serial reaction time test (5-CSRT) performance. Lister Hooded rats received 32-128 mg/kg modafinil and 5-CSRT performance was assessed under standard and test parametric conditions in which the attentional load was increased, and also under conditions of scopolamine pre-treatment. Modafinil failed to significantly enhance 5-CSRT performance under standard conditions. Similarly, modafinil was unable to reverse the deficits in accuracy and/or increased omission errors induced by either parametric or pharmacological manipulations. Indeed, at higher doses, modafinil caused an increase in premature responding under certain test conditions, suggestive of increased impulsivity. The present findings suggest that, although modafinil may enhance vigilance in sleep-deprived human subjects, attentional processes in normal awake rats remain unaffected. No evidence was found to support a modafinil-induced improvement in response control; rather, under conditions of increased attentional load, modafinil appeared to facilitate impulsive responding. Finally, the failure of modafinil to improve a scopolamine-induced performance deficit suggests that modafinil does not act on the cholinergic system directly.

Alpha3IA (6-(4-pyridyl)-5-(4-methoxyphenyl)-3-carbomethoxy-1-methyl-1H-pyridin-2-one) is a pyridone with higher binding and functional affinity and greater inverse agonist efficacy for GABA(A) receptors containing an alpha3 rather than an alpha1, alpha2 or alpha5 subunit. If doses are selected that minimise the occupancy at these latter subtypes, then the in vivo effects of alpha3IA are most probably mediated by the alpha3 subtype. Alpha3IA has good CNS penetration in rats and mice as measured using a [(3)H]Ro 15-1788 in vivo binding assay. At doses in rats that produce relatively low levels of occupancy (12%) in the cerebellum (i.e. alpha1-containing receptors), alpha3IA (30 mg kg(-1) i.p.), like the nonselective partial inverse agonist N-methyl-beta-carboline-3-carboxamide (FG 7142), not only caused behavioural disruption in an operant, chain-pulling assay but was also anxiogenic in the elevated plus maze, an anxiogenic-like effect that could be blocked with the benzodiazepine antagonist Ro 15-1788 (flumazenil). Neurochemically, alpha3IA (30 mg kg(-1) i.p.) as well as FG 7142 (15 mg kg(-1) i.p.) increased the concentration of the dopamine metabolite 3,4-dihydroxyphenylacetic acid in rat medial prefrontal cortex by 74 and 68%, respectively, relative to vehicle-treated animals, a response that mimicked that seen following immobilisation stress. Taken together, these data demonstrate that an inverse agonist selective for GABA(A) receptors containing an alpha3 subunit is anxiogenic, and suggest that since alpha3-containing GABA(A) receptors play a role in anxiety, then agonists selective for this subtype should be anxiolytic.

Benzodiazepines exert their effects by binding to multiple subtypes of the GABAA receptor, the predominant subtypes in the brain being those that contain alpha1-, alpha2-, alpha3-, and alpha5-subunits. To understand the potentially different roles of these subtypes in the therapeutic and side effects of benzodiazepines, we evaluated GABAA receptor subtype-preferring compounds in nonhuman primate models predictive of anxiolytic, sedative, motor, subjective, and reinforcing effects of benzodiazepine-type drugs. These compounds included zolpidem, which shows preferential binding to GABAA receptors containing alpha1-subunits (alpha1GABAA receptors); L-838,417, which shows functional selectivity for alpha2GABAA, alpha3GABAA, and alpha5GABAA receptors; and nonselective conventional benzodiazepines. The results provide evidence in nonhuman primates that alpha1GABAA receptors do not play a key role in the anxiolytic and muscle-relaxant properties of benzodiazepine-type drugs; instead, these effects involve alpha2GABAA, alpha3GABAA, and/or alpha5GABAA subtypes. Our results also suggest that the alpha1GABAA receptor subtype might be critically involved in the subjective, sedative, and motor effects of benzodiazepine-type drugs. In contrast, stimulation of alpha1GABAA receptors is sufficient, but not necessary, for mediation of the abuse potential of these drugs.

Drugs modulating gamma-aminobutyric acid (GABA) transmission via the benzodiazepine (BZ) site on the gamma-aminobutyric acid type A (GABAA) receptor have been in widespread use for more than 40 years to treat anxiety, epilepsy, and sleep disorders. These drugs have been shown to be safe, well tolerated, and effective although the mechanism by they produce a myriad of pharmacologic effects remains elusive. In recent years it has been discovered that, although the GABAA receptor is widely distributed in the brain, the substructure and composition of the receptor differs from between brain regions. Termed "GABAA receptor subtypes" their discovery leads to speculation that different subtypes may mediate specific effects of BZs such as anxiety or sedation. The phenotypic analysis of transgenic knock-in and knock-out mice in which particular GABAA receptors were rendered insensitive to the effects of BZ while others were unaffected confirmed this speculation. Subsequently, subtype-specific GABAA ligands were developed that, for example, retained the anxiolytic effects of BZs but were devoid of their sedative effects. Therefore, it may be possible to develop effective anxiolytic compounds that have a much reduced side-effect profile compared with existing drugs.

(3-tert-Butyl-7-(5-methylisoxazol-3-yl)-2-(1-methyl-1H-1,2,4-triazol-5-ylmethoxy)pyrazolo[1,5-d][1,2,4]triazine (13) has been identified as a functionally selective, inverse agonist at the benzodiazepine site of GABA(A) alpha5 receptors. 13 is orally bioavailable, readily penetrates the CNS, and enhances performance in animal models of cognition. It does not exhibit the convulsant, proconvulsant, or anxiogenic activity associated with nonselective GABA(A) inverse agonists.

A homogeneous time-resolved fluorescence immunoassay for detection of beta-amyloid (A beta) peptides has been adapted for quantification of A beta(40) and A beta(42) accumulation in brains of APP695SWE transgenic mice. These over-express human beta APP(swe), beta-amyloid precursor protein (beta-APP) containing the K670N/M671L 'Swedish' familial Alzheimer's disease (FAD) mutation. Both peptides start to accumulate in this line from about 260 to 280 days of age. Co-expression of a human presenilin-1 (PS1) transgene containing the A246E FAD mutation accelerates deposition and also favors-at least initially-accumulation of A beta(42) so that the A beta(2):A beta(40) ratio of peptides from 7- to 12-month-old APP695SWE x PS1A246E animals is significantly elevated above that observed throughout the lifetime of APP695SWE mice. These findings, supported by parallel immunohistochemical staining and surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry data, offer important longitudinal characterization of two mouse models of cerebral amyloidosis. Application of the same extraction and quantitation procedures to samples of temporal cortex from AD sufferers indicates however that A beta(40) is only a minor component of beta-amyloid in humans.

The i.v. agent etomidate exerts its anaesthetic actions through potentiation of gamma-aminobutyric acid-A receptors containing beta2 and beta3 subunits. It was recently shown that the beta2 subunit contributes to the sedative properties of etomidate, whereas the beta3 subunit is responsible for its anaesthetic properties. However, these studies evaluated anaesthetic effects in point mutation mice in which the effect of etomidate was decreased, but not abolished, at the beta2 subunit. Here we have used beta2 knockout mice to completely remove any contribution of the beta2 subunit to the effects of etomidate. Etomidate was equally anaesthetic in wildtype and knockout mice, thus further confirming that efficacy at the beta3 subunit only is sufficient to induce general anaesthesia.

The identification of a novel series of 7,8,9,10-tetrahydro-(7,10-ethano)-1,2,4-triazolo[3,4-a]phthalazines as GABA(A)alpha5 inverse agonists, which have both binding and functional (efficacy) selectivity for the benzodiazepine binding site of alpha5- over alpha1-, alpha2-, and alpha3-containing GABA(A) receptor subtypes, is described. Binding selectivity was determined to a large part by the degree of planarity of the fused ring system whereas functional selectivity was dependent on the nature of the heterocycle at the 3-position of the triazolopyridazine ring. 3-Furan and 5-methylisoxazole were shown to be optimal for GABA(A)alpha5 functional selectvity. 3-(5-Methylisoxazol-3-yl)-6-(2-pyridyl)methyloxy-1,2,4-triazolo[3,4-a]phthalazine (43) was identified as a full inverse agonist at the GABA(A)alpha5 subtype with functional selectivity over the other GABA(A) receptor subtypes and good oral bioavailability.

Extinction following positively reinforced operant conditioning reduces response frequency, at least in part through the aversive or frustrative effects of non-reinforcement. According to J.A. Gray's theory, non-reinforcement activates the behavioural inhibition system which in turn causes anxiety. As predicted, anxiolytic drugs including benzodiazepines affect the operant extinction process. Recent studies have shown that reducing GABA-mediated neurotransmission retards extinction of aversive conditioning. We have shown in a series of studies that anxiolytic compounds that potentiate GABA facilitate extinction of positively reinforced fixed-ratio operant behaviour in C57B1/6 male mice. This effect does not occur in the early stages of extinction, nor is it dependent on cumulative effects of the compound administered. Potentiation of GABA at later stages has the effect of increasing sensitivity to the extinction contingency and facilitates the inhibition of the behaviour that is no longer required. The GABAergic hypnotic, zolpidem, has the same selective effects on operant extinction in this procedure. The effects of zolpidem are not due to sedative action. There is evidence across our series of experiments that different GABA-A subtype receptors are involved in extinction facilitation and anxiolysis. Consequently, this procedure may not be an appropriate model for anxiolytic drug action, but it may be a useful technique for analysing the neural bases of extinction and designing therapeutic interventions in humans where failure to extinguish inappropriate behaviours can lead to pathological conditions such as post-traumatic stress disorder.

Relatively little is known about the role of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in extinction of appetitively motivated tasks. The benzodiazepine (BZ) chlordiazepoxide (CDP) was administered during extinction and re-acquisition of lever pressing by mice following food reinforced discrete-trial fixed-ratio 5 (FR-5) training. Typical FR behaviour was established during baseline training and persisted for several extinction sessions. There were 15 extinction sessions in all, followed by six re-acquisition sessions where food reinforcement was re-introduced. In a 2x2x2 between-group design, CDP (15 mg/kg) or vehicle injections were given prior to either the last two food reinforcement sessions and the first 10 extinction sessions, or the final five extinction sessions, or the six re-acquisition sessions. Initially CDP had no effect on the rate of extinction, but after several extinction sessions it significantly facilitated it. Surprisingly, if CDP was administered only after several sessions of extinction, it immediately produced facilitation. Thus the delayed effects of CDP are not due to drug accumulation. These data suggest that some neural change must occur before CDP can affect extinction processes. In re-acquisition sessions, CDP facilitated the reinstatement of food-reinforced lever pressing. Implications for neural and behavioural accounts of operant extinction are discussed.

Nonselective inverse agonists at the gamma-aminobutyric acid(A) (GABA-A) benzodiazepine binding site have cognition-enhancing effects in animals but are anxiogenic and can precipitate convulsions. Herein, we describe novel GABA-A alpha5 subtype inverse agonists leading to the identification of 16 as an orally active, functionally selective compound that enhances cognition in animals without anxiogenic or convulsant effects. Compounds of this type may be useful in the symptomatic treatment of memory impairment associated with Alzheimer's disease and related dementias.

Studies with our screening lead 5 and the literature compound 6 led to the identification of 6-benzyloxy-3-(4-methoxy)phenyl-1,2,4-triazolo[3,4-a]phthalazine 8 as a ligand with binding selectivity for the gamma-aminobutyric acid-A (GABA-A) alpha 3- and alpha 5-containing receptor subtypes over the GABA-A alpha 1 subtype (K(i): alpha 2 = 850 nM, alpha 3 = 170 nM, alpha 5 = 72 nM, alpha 1 = 1400 nM). Early optimization studies identified the close analogue 10 (K(i): alpha 2 = 16 nM, alpha 3 = 41 nM, alpha 5 = 38 nM, alpha 1 = 280 nM) as a suitable lead for further study. High-affinity ligands were identified by replacing the 6-benzyloxy group of compound 10 with 2-pyridylmethoxy (compound 29), but binding selectivity was not enhanced (K(i): alpha 2 = 1.7 nM, alpha 3 = 0.71 nM, alpha 5 = 0.33 nM, alpha 1 = 2.7 nM). Furthermore, on evaluation in xenopus oocytes,(22) 29 was discovered to be a weak to moderate inverse agonist at all four receptor subtypes (alpha 1, -7%; alpha 2, -5%; alpha 3, -16%; alpha 5, -5%). Replacement of the 3-phenyl group of 29 with alternatives led to reduced affinity, and smaller 3-substituents led to reduced efficacy. Methyl substitution of the benzo-fused ring of 29 at the 7-, 8-, and 10-positions resulted in increased efficacy although selectivity was abolished. Increased efficacy and retention of selectivity for alpha 3 over alpha 1 was achieved with the 7,8,9,10-tetrahydro-(7,10-ethano)-phthalazine 62. Compound 62 is currently one of the most binding selective GABA-A alpha 3-benzodiazepine-site partial agonists known, and although its selectivity is limited, its good pharmacokinetic profile in the rat (33% oral bioavailability after a 3 mg/kg dose, reaching a peak plasma concentration of 179 ng/mL; half-life of 1 h) made it a useful pharmacological tool to explore the effect of a GABA-A alpha 2/alpha 3 agonist in vivo.

Several recent studies have shown that reducing gamma-aminobutyric acid (GABA)-mediated neurotransmission retards extinction of aversive conditioning. However, relatively little is known about the effect of GABA on extinction of appetitively motivated tasks. We examined the effect of chlordiazepoxide (CDP), a classical benzodiazepine (BZ) and two novel subtype-selective BZs when administered to male C57Bl/6 mice during extinction following training on a discrete-trial fixed-ratio 5 (FR5) food reinforced lever-press procedure. Initially CDP had no effect, but after several extinction sessions CDP significantly facilitated extinction, i.e. slowed responding, compared with vehicle-treated mice. This effect was not due to drug accumulation because mice switched from vehicle treatment to CDP late in extinction showed facilitation immediately. Likewise, this effect could not be attributed to sedation because the dose of CDP used (15 mg/kg i.p.) did not suppress locomotor activity. The two novel subtype-selective BZ partial agonists, L-838417 and TP13, selectively facilitated extinction in similar fashion to CDP. The non-GABAergic anxiolytic buspirone was also tested and found to have similar effects when administered at a non-sedating dose. These studies demonstrate that GABA-mediated processes are important during extinction of an appetitively motivated task, but only after the animals have experienced several extinction sessions.