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NEUROPHARMACOLOGY

Correlating Efficacy in Rodent Cognition Models with in Vivo 5-Hydroxytryptamine_1A Receptor Occupancy by a Novel Antagonist, (R)-N-(2-Methyl-(4-indolyl-1-piperazinyl)ethyl)-N-(2-pyridinyl)-cyclohexane Carboxamide (WAY-101405)

Discovery Neuroscience (W.D.H., T.H.A., S.A., T.A.C., L.A.D., M.D., S.M.G., Z.A.H., H.v.d.L., S.R.-L., D.L.S., K.S., S.J.S.R., L.E.S.) and Chemical and Screening Sciences (W.E.C., B.L.H., M.G.K., A.L.S., M.-Y.Z.), Wyeth Research, Princeton, New Jersey; and Drug Safety and Metabolism, Wyeth Research, Collegeville, Pennsylvania (I.B.F., J.K., C.T.)

Received for publication October 15, 2007
Accepted January 7, 2008.

	Abstract

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5-Hydroxytryptamine (5-HT)_1A receptors play an important role in multiple cognitive processes, and compelling evidence suggests that 5-HT_1A antagonists can reverse cognitive impairment. We have examined the therapeutic potential of a potent (K_i = 1.1 nM), selective (>100-fold), orally bioavailable, silent 5-HT_1A receptor antagonist (K_B = 1.3 nM) (R)-N-(2-methyl-(4-indolyl-1-piperazinyl)-ethyl)-N-(2-pyridinyl)-cyclohexane carboxamide (WAY-101405). Oral administration of WAY-101405 was shown to be effective in multiple rodent models of learning and memory. In a novel object recognition paradigm, 1 mg/kg enhanced retention (memory) for previously learned information, and it was able to reverse the memory deficits induced by scopolamine. WAY-101405 (1 mg/kg) was also able to reverse scopolamine-induced deficits in a rat contextual fear conditioning model. In the Morris water maze, WAY-101405 (3 mg/kg) significantly improved learning in a paradigm of increasing task difficulty. In vivo microdialysis studies in the dorsal hippocampus of freely moving adult rats demonstrated that acute administration of WAY-101405 (10 mg/kg) increased extracellular acetylcholine levels. The selective radioligand [³H]WAY-100635, administered i.v., was used for in vivo receptor occupancy studies, where WAY-101405 occupied 5-HT_1A receptors in the rat cortex, with an ED₅₀ value of 0.1 mg/kg p.o. Taken together, these studies demonstrate that WAY-101405 is a potent and selective, brain penetrant, orally bioavailable 5-HT_1A receptor "silent" antagonist that is effective in preclinical memory paradigms at doses where approximately 90% of the postsynaptic 5-HT_1A receptors are occupied. These results further support the rationale for use of this compound class in the treatment of cognitive dysfunction associated with psychiatric and neurological conditions.

The prototypical 5-HT_1A receptor agonist, 8-OH-DPAT, has been shown to impair learning and memory in a range of rodent models, including water maze, passive avoidance, radial maze, delayed nonmatching to position, and five-choice serial reaction task (for reviews, see Schechter et al., 2002; Myhrer, 2003). More recently, studies in human volunteers have shown that oral administration of the 5-HT_1A receptor agonist tandospirone impaired verbal memory (Yasuno et al., 2003). Furthermore, a significant negative correlation was observed between verbal and general memory indices and the density of 5-HT_1A receptors in the hippocampus as assessed using [¹¹C]WAY-100635 and positron emission tomography (Yasuno et al., 2003), expanding on earlier studies that demonstrated changes in human 5-HT_1A receptor expression in aged subjects and Alzheimer's disease patients (Meltzer et al., 1998; Tauscher et al., 2001).

WAY-100135 was the first selective 5-HT_1A receptor antagonist to be described (Fletcher et al., 1993), but was later shown to have partial agonist activity at higher doses (Assié and Koek, 1996). Subsequently, WAY-100635, an achiral analog of WAY-100135, was synthesized and was shown to be the first selective and silent 5-HT_1A receptor antagonist in a range of pharmacological assays (Forster et al., 1995). Other 5-HT_1A receptor antagonists include NAD-299 (Johansson et al., 1997) and LY-426965 (Rasmussen et al., 2000), which are structural analogs of 8-OH-DPAT and pindolol, respectively. We have recently described the potent and selective 5-HT_1A receptor antagonist lecozotan, which is currently in clinical trials for treatment of the cognitive deficits associated with Alzheimer's disease (Schechter et al., 2005).

These compounds have been evaluated in multiple preclinical studies and although not equivocal, there is considerable data supporting the hypothesis that 5-HT_1A receptor antagonists improve performance in cognitive tasks. For example, WAY-100135 and WAY-100635 attenuated the impairment of spatial learning caused by the administration of intrahippocampal scopolamine, 7-chloro-kynurenic acid, or a competitive NMDA receptor antagonist, MK-801, in rats (reviewed by Schechter et al., 2002). WAY-100635 reverses the choice accuracy deficit of a rat whose nucleus basalis magnocellularis had been lesioned, but it does not shorten correct response latencies (Balducci et al., 2003). WAY-100635 reversed 8-OH-DPAT-induced deficits in a rat recognition memory task (Pitsikas et al., 2003). WAY-100635 and NAD-299 have been evaluated in water maze and passive avoidance in rats (Lüttgen et al., 2005) and passive avoidance in mice (Madjid et al., 2006), and they have shown procognitive effects.

Studies have also been described in nonhuman primates. WAY-100635 and lecozotan reversed the cognitive deficits induced by a fornix lesion (Harder et al., 1996) or specific cholinergic lesions of the hippocampus (Schechter et al., 2005) and deficits induced by the N-methyl-D-aspartate receptor antagonist MK-801 (Schechter et al., 2005) in marmosets. In aged rhesus monkeys, lecozotan produced a significant improvement in task performance efficiency (Schechter et al., 2005).

Although WAY-100635 has been used extensively in determining the consequences of 5-HT_1A receptor blockade, it is not orally active and it has a relatively short half-life that has precluded its clinical development (Childers et al., 2005; unpublished data). In an attempt to resolve these issues, WAY-101405, an indole piperazine derivative of WAY-100635, was synthesized. Here, we describe the in vitro pharmacological characterization of WAY-101405, a novel 5-HT_1A receptor antagonist. Pharmacokinetic, pharmacodynamic, and in vivo receptor occupancy were used to demonstrate that WAY-101405 is orally bioavailable, brain-penetrant, and has a pharmacokinetic profile that is superior to WAY-100635, the most commonly used reference compound. In addition, further supporting evidence for the role of 5-HT_1A receptors in cognitive function is provided from these experiments investigating the effects of WAY-101405 in multiple rat cognitive paradigms, and we have measured in vivo receptor occupancy at efficacious doses. These results further support the rationale for use of this compound class in the treatment of cognitive dysfunction associated with psychiatric and neurological conditions.

	Materials and Methods

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Animals. Adult male Sprague-Dawley rats (225–350 g; Charles River Laboratories, Inc., Wilmington, MA) were used in the pharmacokinetic, in vivo microdialysis, and in vivo receptor occupancy studies. Adult male Long-Evans rats (180–300 g; Charles River Laboratories, Inc.) were used in the novel object recognition, contextual fear conditioning, and water maze studies. All animals were group-housed, except where noted, in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility that was maintained on a 12-h light/dark cycle (lights on at 6:00 AM). Food and water were available ad libitum, except where noted. All studies were previously approved by the Institutional Animal Care and Use Committee, and they were performed in accordance with the Principles of Laboratory Animal Care as adopted and promulgated by the National Institutes of Health (1996).

Drugs. WAY-101405 and donepezil (Aricept) were prepared by Wyeth Research (Princeton, NJ). 5-HT, 8-OH-DPAT, scopolamine, Tween 80, and methylcellulose were purchased from Sigma-Aldrich (St. Louis, MO). [³H]8-OH-DPAT (200–240 Ci/mmol) and [³H]WAY-100635 (60–86 Ci/mmol) were purchased from GE Healthcare (Piscataway, NJ). All other materials were analytical grade, and they were obtained from Aldrich Chemical Co. (Milwaukee, WI) and Sigma-Aldrich unless otherwise stated. Scopolamine and donepezil were dissolved in sterile saline vehicle for i.p. administration. 8-OH-DPAT and WAY-101405 were dissolved in sterile saline vehicle for s.c. administration (in vivo microdialysis measurement of 5-HT levels). WAY-101405 was dissolved in water for pharmacokinetic studies (i.v. and p.o. administration). WAY-101405 was suspended in a 2% Tween 80/0.5% methylcellulose solution, and it was administered p.o. for all other studies. All solutions were administered at a volume of 1 ml/kg, and dose calculations were based on the active moiety.

5-HT_1A Receptor Cloning and Preparation of Membranes. Stable Chinese hamster ovary cell lines expressing the human 5-HT_1A receptor subtype (h5-HT_1A CHO cells) or rat 5-HT_1A receptor subtype (r5-HT_1A CHO cells) were used throughout this study; the generation of these cell lines has been described previously (Schechter et al., 2005). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, non-essential amino acids, and penicillin/streptomycin. Cells were grown to 95 to 100% confluence as a monolayer before membranes were harvested for binding studies. Cells were gently scraped from the culture plates, they were transferred to centrifuge tubes, and then they were washed twice by centrifugation (2000 rpm for 10 min at 4°C) in buffer (50 mM Tris-HCl, pH 7.5). The resulting pellets were aliquoted, and then they were stored at –80°C. For preparation of the membranes, cell pellets were homogenized in ice-cold 50 mM Tris-HCl buffer, pH 7.5, for approximately 20 s using an Ultra-Turrax homogenizer (IKA Works, Inc., Wilmington, DE). The homogenates were centrifuged at 40,000g for 20 min at 4°C, and the resulting pellet was rehomogenized and centrifuged as described above. The membranes were finally resuspended in the same buffer at a concentration of approximately 5 mg/ml, and then they were stored at –80°C until use. On the day of assay, the membranes were thawed on ice and resuspended in buffer.

Radioligand Binding Assays. 5-HT_1A receptor binding studies were carried out in membranes derived from h5-HT_1A and r5-HT_1A CHO cells in 50 mM Tris-HCl, pH 7.4, in a 96-well microtiter plates in a total volume of 250 µl. [³H]8-OH-DPAT was used at a final concentration of 1.5 nM in competition binding studies, and nonspecific binding was defined with 10 µM 5-HT. The binding assays were initiated by the addition of 50 µl of the 5-HT_1A cell membranes (0.05 mg protein/sample), and they were incubated at 25°C for 30 min. The reaction was terminated by vacuum filtration through presoaked (0.5% polyethylenimine) Unifilter GF/B filter plates (PerkinElmer Life and Analytical Sciences, Boston, MA) using a Packard Filtermate 96 cell harvester (PerkinElmer Life and Analytical Sciences). Microscint-20 scintillation cocktail (PerkinElmer Life and Analytical Sciences) was added, and radioactivity was measured by liquid scintillation spectrometry using a Packard TopCount liquid scintillation counter (PerkinElmer Life and Analytical Sciences). Protein concentrations were determined by the method of Bradford (1976) using bovine serum albumin as the standard. Measurements were taken at 595 nm with a Pharmacia Biotech Ultraspec 3000 spectrophotometer (Pharmacia Biotech, Cambridge, UK). Binding data were analyzed by GraphPad Prism (GraphPad Software Inc., San Diego, CA), a computer-assisted nonlinear regression analysis program, to determine the concentration of drug inhibiting specific radioligand binding by 50% (IC₅₀). K_i values were then calculated from the IC₅₀ value, as described previously (Hirst et al., 2003), using K_D values determined in saturation studies.

WAY-101405 was profiled in a commercially available panel of 61 neurotransmitter receptors, transporters, ion channels, and enzymes sites (NovaScreen, Hannover, MD). Competition binding at these sites was determined at three concentrations of WAY-101405 (1, 100, and 10,000 nM). A known reference compound was tested at each site as a positive control. Further details of the methodologies for the assays can be found at http://www.novascreen.com.

cAMP Accumulation Assays. Intracellular cAMP levels were measured in h5-HT_1A CHO cells, grown in 96-well plates. Cells were preincubated at 37°C for 15 min in Krebs' solution. Functional activity of WAY-101405 was assessed by treating the cells with forskolin (10 µM final concentration) followed immediately by the test compound at concentrations ranging from 0.1 to 10,000 nM. Then, cells were incubated for an additional 10 min at 37°C. An apparent K_B value was determined for WAY-101405 in the presence of 10 µM forskolin, 10 nM 8-OH-DPAT plus increasing concentrations of the antagonist (as described above). Plates were incubated for 10 min at 37°C, and the assay was terminated by the addition of 0.5 M perchloric acid. Plates were stored at –20°C before the assessment of cAMP formation by a cAMP scintillation proximity assay (GE Healthcare). cAMP accumulation data were generated in singlet within each experiment, and each experiment was carried out at least three times. Curve fitting of the mean data were generated by a four-parameter logistic equation using Prism (GraphPad Software Inc.) to determine the IC₅₀ value. An apparent K_B value was determined using the Gaddum equation, using concentration ratios calculated from the IC₅₀ values from each individual curve (Hirst et al., 2003).

Pharmacokinetic Studies in the Rat. The pharmacokinetics of WAY-101405 were characterized in fasted (overnight) male Sprague-Dawley rats (250–300 g) after a single i.v. bolus dose of 1 mg/kg and a single oral dose of 10 mg/kg. Serial blood samples were collected at intervals up to 8 h after WAY-101405 administration into tubes containing EDTA as anticoagulant, and the contents was diluted 1:1 with deionized water. In a separate study, brain tissue was removed after an oral administration of WAY-101405 (0.3 mg/kg), it was washed free of blood in sterile saline, and it was homogenized in deionized water [50:50 (v/v)]. Samples were extracted using protein precipitation with acetonitrile containing an internal standard. The samples were vortexed, and then they were centrifuged (3400 rpm for 5 min), and the supernatant was evaporated under an N₂ atmosphere at 37°C. The samples were reconstituted in acetonitrile/deionized water [50:50 (v/v)], and the they were analyzed for WAY-101405 concentration by reverse phase high-performance liquid chromatography (HPLC)-tandem mass spectrometry by using a heat-assisted electrospray interface in positive ion mode. The lower limit of quantification was 0.1 ng/ml, using a 50-µl aliquot of plasma. Noncompartmental pharmacokinetic parameters were obtained from the blood concentration-time curves using WinNonlin Professional version 3.3 (Pharsight, Mountain View, CA). Oral bioavailability was calculated as the ratio of the area under the blood concentration versus time curve after p.o. and i.v. doses after normalizing for dose.

In Vivo Microdialysis. Microdialysis studies were carried out in adult male Sprague-Dawley rats (280–350 g) at the time of surgery. After induction of anesthesia with 3% halothane (Fluothane; Zeneca, Cheshire, UK), animals were secured in a stereotaxic frame with ear and incisor bars (David Kopf, Tujunga, CA). Anesthesia was maintained by continuous administration of halothane (1–2%) while a microdialysis guide cannula (CMA/12; CMA/Microdialysis, Stockholm, Sweden) was implanted above the dorsal hippocampus. Coordinates for the hippocampus (RC, –4.8; L, –5.0; and V, –4.4) were taken according to Paxinos and Watson (1986). The guide cannula was secured to the skull using dental acrylic (Plastics One, Roanoke, VA) and two small stainless steel screws (Plastics One). A s.c. cannula was also implanted at this time between the animal's shoulders. After surgery, animals were individually housed in Plexiglas cages (45 x 45 x 30 cm), with free access to food and water. The following day, rats were used in microdialysis experiments; all experiments were performed during the light phase. Microdialysis probes (CMA12 14/02; CMA/Microdialysis) were equilibrated according to manufacturer's specifications. Microdialysis probes were perfused with artificial cerebrospinal fluid (125 mM NaCl, 3 mM KCl, 0.75 mM MgSO₄, and 1.2 mM CaCl₂, pH 7.4) before insertion in the guide cannula. The microdialysis probe was then inserted into the guide cannula, and it was perfused with artificial cerebrospinal fluid at a flow rate of 1 µl/min. A 3-h stabilization period was allowed following probe insertion before dialysate sampling was initiated. Samples were collected every 20 min for 5-HT samples and every 40 min for acetylcholine samples. 5-HT levels were determined in four control samples, taken before drug injection, to achieve a steady baseline. These samples were averaged, and all subsequent values were expressed as a percentage of this preinjection value. Vehicle, WAY-101405, or 8-OH-DPAT was injected (n = 6–14/treatment group), via the s.c. cannula, after preinjection baseline determination of 5-HT levels. Acetylcholine levels were determined in three control samples, taken before WAY-101405 administration (10 mg/kg p.o.), to achieve a steady baseline (n = 4–7/treatment group). Acetylcholine levels were expressed as a percentage of vehicle controls. For acetylcholine analysis, samples were immediately frozen on dry ice after collection. At the end of each experiment, animals were euthanized, and the probe placement was verified histologically. Data from animals with incorrect probe placement were discarded.

5-HT was separated by reverse phase HPLC (C18 ODS3 column, 150 x 3.0 mm; Metachem, Torrance, CA), and it was detected using an Antec electrochemical detector (Antec, Rotterdam, The Netherlands) set at a potential of 0.65 V versus a silver/silver chloride reference electrode. Mobile phase was delivered by a Jasco PU980 HPLC pump (Jasco Ltd., Essex, UK) at 0.5 ml/min, and the phase contained 0.15 M NaH₂PO₄ buffer, pH 4.6, 0.25 mM EDTA, 1.5 mM 1-octane sodium sulfonic acid, and 10% methanol/2% isopropanol. Data were acquired using the Atlas software package (Thermo Electron Corporation, Waltham, MA). The femtomole perfusate values of 5-HT for the first four baseline samples were averaged, and this value is denoted as 100%. Subsequent sample values were expressed as a percentage of this preinjection control value. All results were analyzed by two-way analysis of variance (ANOVA) with repeated measures followed by pairwise comparisons using Bonferroni adjustment for multiple comparisons using the StatView software application (Abacus Concepts, Berkeley, CA) for the PC.

Dialysate levels of acetylcholine were determined using liquid chromatography-tandem mass spectrometry as described previously (Zhang et al., 2007). In brief, separation of acetylcholine was performed on a Supelco LC-SCX 5-µm HPLC column (2.1 x 150 mm) (Supelco, Bellefonte, PA). Mobile phase A was composed of 60, 20, and 20% (v/v) H₂O, buffer (79.5 mM ammonium acetate and 63.5 mM ammonium formate, pH 4.0), and acetonitrile, respectively. Mobile phase B was composed of 20 and 80% (v/v) buffer (79.5 mM ammonium acetate and 63.5 mM ammonium formate, pH 4.0), and acetonitrile, respectively. Eluates were detected using a Micromass spectrometer (Waters, Milford, MA) coupled with HP 1100 (Agilent Technologies, Palo Alto, CA) operated in positive ion mode and monitored with molecular reaction monitoring, with parent ion m/z 146.1 and product ion m/z 87.1. Data were quantified using peak area against an internal standard, and they were acquired using MassLynx software (Micromass, Beverly, MA). Acetylcholine levels were expressed as a percentage of vehicle controls. All results were analyzed by two-way ANOVA with repeated measures followed by pairwise comparisons using Bonferroni adjustment for multiple comparisons using SAS version 1.03 (SAS Institute, Cary, NC) within Excel (Microsoft, Redmond, WA).

Novel Object Recognition. Male Long-Evans rats (180–200 g), housed individually, were used in the novel object recognition studies. This model is based on the greater spontaneous exploration of a novel object, compared with a familiar object, shown by rodents (Ennaceur and Delacour, 1988). Novel object recognition training and testing were performed in a circular field (70 cm in diameter and 30 cm in height) constructed out of plastic and containing soiled bedding. The field was surrounded by black curtains to mask extrafield cues, and was located in a dimly lit room (10 lux at the level of the area) in the presence of white noise (65 dB). Animal performance was tracked by video, and it was monitored by an experimenter located outside of the testing room. Objects, constructed with Duplo (Lego) blocks, were placed on the arena floor in one of four locations spaced evenly around the field and approximately 10 cm from the field's edge. To avoid possible olfactory cues, multiple copies of the objects were used throughout the study, and they were cleaned with a 30% ethanol solution between animal tests. Rats displayed no preference or aversion to either of the objects, and they spent an equivalent amount of time exploring objects if both were presented simultaneously (data not shown). The visual recognition task was divided into three sessions: habituation, a sample trial, and a choice trial. During habituation the animals were placed into the field containing two identical yellow cubes (10 x 10 x 10 cm), and they were allowed to explore the field for 15 min. After habituation, rats were returned to their home cage. One day after habituation, animals (n = 10/treatment group) were dosed with drug, and the sample trial was initiated. During the sample trial, rats were allowed to explore the field, now containing two new, identical objects located at opposing compass points, for 5 min. The amount of time exploring the objects was recorded for the entire trial. Exploration was defined as orientation toward the object with the nose of the rat within <2 cm of the object. After the sample trial, rats were returned to their home cages for a 48-h interval for the delay model or a 1-h interval for the scopolamine-induced deficit model. Animals were then tested in the choice trial for recognition memory. The choice trial consisted of a 5-min exploration of the field containing both a familiar, previously explored, object and a novel object; contact time was recorded by an investigator. The location of the objects, counterbalanced across treatment groups, remained constant for each animal during the habituation, sample, and choice trials. The effect of treatment on object exploration during the sample trial was examined using a one-way ANOVA on total contact time followed by Fisher's LSD group mean pairwise comparisons (SAS version 1.03 within Excel). The amount of time exploring the novel and familiar objects across treatment groups was analyzed using a two-factor ANOVA (object type by treatment) using the same statistical package. Planned comparisons in novel versus familiar object exploration within treatment group were evaluated by Fisher's LSD test. Significantly more time spent exploring the novel object than the familiar object during the choice trial represents intact recognition memory for that treatment group.

Contextual Fear Conditioning. Male, Long-Evans rats (200–250 g) were used for contextual fear conditioning studies. Contextual fear conditioning was performed in rat operant chambers (MED Associates, St. Albans, VT) equipped with a grid floor coupled to a current generator, houselight, stimulus lights, and a tone generator. Training and testing procedures were controlled by a computer using MED-PC software (MED Associates). The chambers were located in a sound-isolated room in the presence of red light. Rats (n = 8/treatment group) were trained and tested on two consecutive days. Training consisted of placing a subject in a chamber, illuminating the stimulus and houselights, and then allowing exploration by the rat for 2 min. An auditory tone was presented for 30 s, and the footshock (1.0 mA) was administered for the final 2 s of the tone presentation and coterminated with the tone. This procedure was repeated, with the rats remaining in the chamber before the second tone and footshock, the rats were removed from the chamber 30 s after the second footshock and returned to the homecage. Testing occurred approximately 18 to 20 h after training. A subject was returned to the same chamber in which training occurred, and freezing behavior, in the absence of the auditory cue (tone), was recorded by the experimenter using time sampling (scoring at 10-s intervals for 5 min). Freezing was defined as lack of movement except that required for respiration. Freezing score was converted to a percentage of the maximum number of samples (30), and it was analyzed using a one-way ANOVA followed by Fisher's LSD post-hoc pairwise comparisons using SAS version 1.03 within Excel. High freezing scores indicate memory for the aversive event (no freezing is observed during testing in rats that had been through the training procedure but did not receive a shock). The muscarinic antagonist scopolamine (0.56 mg/kg i.p.), administered 30 min before training, impairs memory for the association during the test session.

Water Maze. Male Long-Evans rats (200–300 g) were used for the water maze studies using the Atlantis protocol (Day and Langston, 2006). The Atlantis water maze is an animal model designed to detect enhancements of spatial memory encoding and retention. A 1.5-m diameter white fiberglass pool with a depth of 0.6 m was used. The pool was raised 0.65 m standing 1.5 m in height from the floor, and four 500-W spotlights were placed in each corner of the room approximately 1 m above the floor facing upward to provide lighting. The pool was filled with water, to a depth of 0.3 m. The pool was made opaque with the addition of 150 ml of nontoxic tempura paint (Michaels, The Arts and Craft Store, Irving, TX). The temperature of the water was 25°C at the beginning of each testing period. The pool was situated in a room that contained a variety of two- and three-dimensional distal cues that provide navigational cues for the animals attempting to locate the hidden platform. The swim paths of the rats were tracked using a video camera suspended centrally above the pool, and all sessions were recorded on videotape. Data were collected and analyzed online using Actimetrics water maze software (Actimetrics, Wilmette, IL).

The design of the experiment is illustrated in Fig. 6a. Vehicle or WAY-101405 (0.3 or 3 mg/kg p.o.) was administered 2 h before trial 1 on each of the four training days (n = 10/treatment group). Animals were also dosed, once daily, during the retention interval, but they did not receive WAY-101405 during the retention tests on day 10.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effects of WAY-101405 in a water maze test. a, rats were pretrained using a visual cue in four consecutive training trials. Rats were then trained for 4 days after receiving vehicle or WAY-101405 (0.3 or 3 mg/kg p.o.) 2 h before trial 1 on each of the four training days. Animals were also dosed, once daily, during the retention interval, but they did not receive WAY-101405 during the retention tests on day 10. b, acquisition curves across four consecutive training trials on days 1 to 4 after treatment with vehicle or WAY-101405 (0.3 or 3 mg/kg p.o.). Data are expressed as mean ± S.E.M. of the escape latency (seconds) of 10 rats per treatment group (***, p < 0.001 versus vehicle group). c, retention tests measured percentage of time in a 32-cm zone around the center of the previously targeted area relative to the corresponding areas in the other three pool quadrants was calculated during the 30-s probe trial length. Data are expressed as mean ± S.E.M. of the percentage of time in target zone of 10 rats per treatment group (***, p < 0.001 versus vehicle group).

After visual cue training, all rats received 4 days of spatial training (four trials/day) with the curtains removed and the distal extramaze cues visible. Each rat was placed in the pool facing the wall at one of the start locations (north, east, south, and west).

The escape platform was located in the southwest quadrant of the water maze, and the location of the platform position remained constant as in standard spatial reference memory water maze procedures (Morris et al., 1982). Using the Atlantis platform (15 cm in diameter, pneumatically controlled submerged platform), rats were trained to dwell within a 35-cm-diameter zone above the platform location for a predetermined time. This activated the platform that was then automatically released from the bottom of the pool allowing the rat to escape on to it (Day and Langston, 2006). The Atlantis platform was set to be activated after 1-s dwell time on day 1, 2 s on day 2, and 3 s on days 3 and 4. Rats were given a maximum of 120 s to find the platform in all trials. If the rat had failed to activate the platform within 90 s, the platform would rise. Once on the platform, rats remained there for 30 s before being removed by the experimenter. Escape latencies were defined as the amount of time taken for the rat to mount the hidden platform, and they were expressed as a mean for each trial on each day. These values represent a measure of spatial memory encoding. The presence of a significant difference between treatments was determined by a two-way (treatment by trial) repeated measures ANOVA for each day (days 1–4) followed by Dunnett's post-hoc comparison using SAS version 1.03 within Excel.

Five days after completion of the last acquisition session, rats were returned to the maze, and they were placed in the tank for a 60-s trial. The escape platform was not accessible to the animals, and their swim paths were tracked throughout this test. Percentage of time in a 52-cm-diameter zone around the center of the previously targeted area relative to the corresponding areas in the other three pool quadrants was calculated during the 60-s probe trial length. The presence of a significant difference between treatments was determined by one-way ANOVA followed by Dunnett's post-hoc comparison using SAS version 1.03 within Excel.

In Vivo Receptor Occupancy. Male Sprague-Dawley rats (225–300 g) were used for the in vivo receptor occupancy studies. Rats received vehicle or WAY-101405 (0.1, 0.3, 1, or 3 mg/kg p.o.) 90 min before administration of [³H]WAY-100635 (7.5 µCi/rat in a volume of 0.3 ml of sterile saline) injected through a lateral tail vein 30 min before sacrifice (n = 4/treatment group). Brains were removed and placed on ice. Frontal cortex, hippocampus, and cerebellum were dissected and weighed. NCS Tissue Solubilizer (GE Healthcare) was added to each sample at a concentration of 1 ml of solvent/100 mg of tissue, and each sample was allowed to solubilize overnight while gently shaking at 50°C. After approximately 16 h of shaking, 30 µlof glacial acetic acid was added per 1 ml of solubilized tissue to minimize background interference. The samples were then allowed to cool. After cooling, an equal volume was removed from each solubilized tissue sample, and then it was transferred to a scintillation vial containing 15 ml of Opti-Fluor (PerkinElmer Life and Analytical Sciences), and the tritium content was determined by liquid scintillation spectrometry using a Canberra-Packard Tri-Carb 2200 liquid scintillation counter (PerkinElmer Life and Analytical Sciences). The 5-HT_1A binding potential (BP) was determined for the frontal cortex and hippocampus in each rat using the cerebellum, a brain region with relatively few 5-HT_1A receptors (Chalmers and Watson, 1991) as a reference tissue for the nonspecific binding of [³H]WAY-100635. Thus, the BP is calculated as (dpm in frontal cortex–dpm in cerebellum)/dpm in cerebellum (Wadenberg et al., 2000). The BP values for the vehicle-treated control group were pooled, and the occupancy for each brain region in each rat was then determined using the following formula: percentage of receptor occupancy = 100 x ((5-HT_1A control–5-HT_1A individual)/5-HT_1A BP control) (Wadenberg et al., 2000).

	Results

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In Vitro Characterization of WAY-101405. The chemical structure of WAY-101405 is shown in Fig. 1. Radioligand binding studies with [³H]8-OH-DPAT demonstrated that WAY-101405 had high affinity for both human and rat 5-HT_1A receptors, with K_i values of 1.13 ± 0.23 and 1.32 ± 0.22 nM, respectively (Table 1). WAY-101405 was selective for the 5-HT_1A receptor over other monoamine receptors that have previously been shown to have affinity for the arylpiperazine pharmacophore (Childers et al., 2005). A Nova-Screen profile of 61 neurotransmitter receptors, transporters, ion channels, and enzymes sites revealed that greater than 50% displacement was only recorded at the highest concentration of 10,000 nM at 13 sites, indicating that WAY-101405 demonstrates significant selectivity for the 5-HT_1A receptor. At a concentration of 100 nM, WAY-101405 did not exhibit significant activity (>50% inhibition) at any of the 61 binding sites. It is noteworthy that the compound was not active at various -adrenergic (1A, 1B, 2A, and 2B), β-adrenergic (1 and 2), adenosine (1 and 2), dopamine (2, 3, and 4), histamine (1, 2, and 3), muscarinic acetylcholine (1, 2, and 3), or serotonin (1B, 1D, 2A, 2C, 3, 4, 5, 6, and 7) receptors.

View larger version (6K): [in this window] [in a new window]   Fig. 1. Chemical structure of WAY-101405.

In functional assays with CHO cells expressing the human 5-HT_1A receptor, WAY-101405 antagonized the 8-OH-DPAT inhibition of forskolin-stimulated cAMP production. In this study, the response to 10 nM 8-OH-DPAT, representing a submaximal concentration, was evaluated in the presence of increasing concentrations of antagonist. The calculated IC₅₀ value for WAY-101405 was 8.81 ± 0.74 nM, which corresponds to an apparent K_B value of 1.27 ± 0.30 nM (Table 1). WAY-101405 alone did not exhibit any agonist activity under any assay conditions.

Pharmacokinetics of WAY-101405. The pharmacokinetics of WAY-101405 were evaluated in male Sprague-Dawley rats. Following single i.v. bolus doses of 1 mg/kg to the rats, WAY-101405 had a high blood clearance of 3.9 l/h/kg (ca. 70% of liver blood flow), with a volume of distribution at steady state of 3.8 l/kg, indicating moderate distribution into tissues, and an apparent half-life of 0.8 h (Table 2). After oral administration of 10 mg/kg, peak blood concentrations of 1640 ng/ml (3.4 µM) were achieved 15 min after dosing, with an oral half-life of 3.1 h. The oral bioavailability of WAY-101405 in the rat was 22% (Table 2). The brain/plasma ratio for WAY-101405 was 5:1, with peak brain levels detected at 15 min and maintained for an additional 2 to 4 h. At 2 h, the total plasma and brain concentrations were 1020 ng/ml and 4015 ng/g, respectively.

In Vivo Microdialysis to Determine Intrinsic Activity. WAY-101405 (1 mg/kg s.c.), administered alone, had no effect on basal levels of 5-HT in the hippocampus of conscious rats compared with vehicle-treated animals (Fig. 2). Treatment with 8-OH-DPAT (0.3 mg/kg s.c.) induced a significant [F(5, 44) = 6.0; p < 0.05] decrease in extracellular 5-HT levels. Pretreatment with 0.1 and 1 mg/kg, WAY-101405 prevented the decrease in 5-HT caused by 8-OH-DPAT. The lower dose, 0.01 mg/kg, of WAY-101405 did not block the 8-OH-DPAT-mediated decreases in extracellular 5-HT levels (Fig. 2).

View larger version (21K): [in this window] [in a new window]   Fig. 2. WAY-101405 blocks an 8-OH-DPAT-mediated decrease of hippocampal 5-HT, measured by in vivo microdialysis. 8-OH-DPAT administered s.c. significantly (p < 0.05) decreased hippocampal extracellular 5-HT levels. Administration of WAY-101405 (1 mg/kg s.c.) alone had no effect on 5-HT. Pretreatment with WAY-101405 (0.1 and 1 mg/kg s.c.) prevented the decrease in 5-HT caused by 8-OH-DPAT. Data are expressed as mean ± S.E.M. of a percentage of preinjection baseline values from 14 rats (vehicle), 6 rats (WAY-101405), 7 rats (WAY-101405 plus 8-OH-DPAT), and 8 rats (8-OH-DPAT) (*, p < 0.05). Baseline 5-HT levels were 42.8 ± 2.7 fmol/20 µl.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effects of WAY-101405 (0.3–10 mg/kg p.o., administered 2 h before training on day 1) on a temporal-induced deficit (48 h) (a) and a scopolamine-induced deficit (0.56 mg/kg i.p., administered 30 min before training on day 1) (b) in a rat novel object recognition paradigm. Mean time (seconds) ± S.E.M. spent exploring novel and familiar objects is shown from 10 rats per treatment group (*, p < 0.05 versus familiar).

In a variation of the novel object recognition paradigm, pharmacological deficits were induced in the rats using the muscarinic antagonist scopolamine (0.56 mg/kg i.p., administered 30 min before training on day 1). The scopolamine pretreatment blocked the retention as demonstrated by the lack of significant difference in exploration of the familiar and novel objects during the choice trial (Fig. 3b). Evidence of significant retention was observed in the vehicle-treated animals and in rats treated with the groups that had received 1 and 3 mg/kg WAY-101405, but not the 0.3-mg/kg group [F(4, 40) = 3.28; p = 0.0204; post-hoc analysis (Fisher's LSD); p < 0.05]. Neither scopolamine alone nor in combination with any of the doses of WAY-101405 altered the exploration of the objects during the sample trial, suggesting that the deficit and improvement were not due to general changes in exploration of the objects (data not shown). Taken together, the data from the novel object recognition models demonstrated a minimal effective dose in these paradigms of 1 mg/kg.

View larger version (20K): [in this window] [in a new window]   Fig. 4. a, donepezil (0.5 mg/kg i.p.) and WAY-101405 (0.3 mg/kg p.o.) do not affect exploration time in trial 1 of the rat novel object recognition paradigm. b, effects of donepezil (0.5 mg/kg i.p., administered 30 min before training on day 1) and WAY-101405 (0.3 mg/kg p.o., administered 2 h before training on day 1) alone and in combination on a temporal induced deficit (48 h) in a rat novel object recognition paradigm. Mean time (seconds) ± S.E.M. spent exploring novel and familiar objects is shown from 10 rats per treatment group (*, p < 0.05 versus familiar).

Contextual learning involves the association of an aversive stimulus (footshock) with a specific cage environment in which the shock occurred (context). Memory for the conditioning is expressed as a context-dependent freezing behavior of the rat in the absence of the shock. Scopolamine (0.56 mg/kg i.p.), administered 30 min before training on day 1, caused a robust deficit in the amount of time the rats spent freezing when reintroduced to the chamber where the training had occurred the previous day (Fig. 5). Clinically effective cognitive enhancers such as donepezil (1 mg/kg i.p.) block this scopolamine-induced disruption of memory (data not shown). WAY-101405 (3 mg/kg p.o., administered 2 h before training) significantly [F(4, 35) = 4.902; p = 0.003; post-hoc analysis (Fisher's LSD); p < 0.05] reversed the scopolamine-induced memory deficits (Fig. 5).

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effects of WAY-101405 (0.3–3 mg/kg p.o., administered 2 h before training on day 1) on a scopolamine-induced deficit (0.56 mg/kg i.p.) in a rat contextual fear conditioning model. Data are expressed as mean ± S.E.M. of the percentage of time spent freezing of eight rats per treatment group (*, p < 0.05 versus vehicle/vehicle group; #, p < 0.05 versus scopolamine/vehicle group).

There was no significant effect of drug treatment on swim speed (data not shown). The retention of this memory was assessed in a drug-free probe test, conducted 5 days after the conclusion of acquisition training, in which animals were placed back in the water maze but without an escape platform. Statistical analysis of the probe data produced a significant treatment zone interaction [F(6, 81) = 3.11; p = 0.0086], with post-hoc analysis (Dunnett's) showing that rats treated with 3 mg/kg WAY-101405 spent a significantly (p < 0.001) greater percentage of time in the previously targeted zone than vehicle- and 0.3 mg/kg-treated animals (Fig. 6c).

In Vivo Microdialysis to Determine Acetylcholine Levels. WAY-101405 (10 mg/kg p.o.) caused a moderate, but statistically significant [F(1,4) = 6.05; p < 0.05], increase in acetylcholine in the rat hippocampus that was maintained for 2 h (Fig. 7).

View larger version (10K): [in this window] [in a new window]   Fig. 7. Temporal effects of WAY-101405 (10 mg/kg p.o.) on extracellular acetylcholine levels in the rat hippocampus. Data are expressed as mean ± S.E.M. of acetylcholine levels expressed as a percentage of vehicle controls from seven rats per vehicle group and four rats in WAY-101405 treatment group (*, p < 0.05 versus vehicle group). Baseline acetylcholine levels were 0.56 ± 0.03 ng/ml (vehicle group) and 0.67 ± 0.07 ng/ml (WAY-101405 group).

View larger version (10K): [in this window] [in a new window]   Fig. 8. Rat brain 5-HT1A receptor occupancy 2 h after oral administration of WAY-101405 (0.1–3 mg/kg). Receptor occupancy, measured by [3H]WAY-100635 in vivo binding assay, increases as a function of dose. [3H]WAY-100635 (7.5 µCi) was injected 30 min before sacrifice. Data are expressed as mean ± S.E.M. of receptor occupancy in frontal cortex and hippocampus using cerebellum as a reference tissue for nonspecific binding from four rats per treatment group.

	Discussion

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WAY-101405 is a novel indole piperazine derivative of WAY-100635, which has been extensively characterized in this report. This compound was shown to have nanomolar affinity for human and rat 5-HT_1A receptor in radioligand binding studies, and it was greater than 100-fold selective over all other neurotransmitter receptors, transporters, ion channels, and enzymes sites tested to date, including dopamine D₄ receptors, which have recently been shown to be a potential binding site for WAY-100635 (Chemel et al., 2006). In vitro functional studies demonstrated that the compound was a potent antagonist, with no evidence of intrinsic activity.

The lack of oral bioavailability seen with WAY-100635 limited its clinical potential (Childers et al., 2005), and this was a key parameter in the optimization of the physical and pharmacological properties of subsequent molecules that lead to the identification of WAY-101405. Pharmacokinetic evaluation of WAY-101405 demonstrated oral bioavailability of 22% and a brain/plasma ratio of 5:1, with peak brain levels detected at 15 min and maintained for an additional 2 to 4 h. Previously determined pharmacodynamic effects, in fixed ratio operant response studies in rats (unpublished observations), showed a good correlation with the pharmacokinetic data reported in the present study, and these combined results determined the 2-h pretreatment time that was used in the cognitive assays and receptor occupancy studies.

The estimation of the intrinsic activity of 5-HT_1A receptor ligands is pivotal in characterizing the pharmacological and potential therapeutic utility of a specific compound of this class. Central 5-HT_1A receptors exist both in presynaptic (somatodendritic autoreceptor) and postsynaptic locations within the central nervous system, and they differ in their functional response to partial agonists (Hoyer et al., 2002). Activation of the presynaptic receptor on the raphe nuclei reduces both the rate of firing of serotonin neurons and the corresponding release of 5-HT from the nerve terminals. Traditionally, physiological activity mediated by the somatodendritic 5-HT_1A autoreceptors localized on the cell bodies in the midbrain have been measured by electrophysiological techniques and neurochemical techniques such as in vivo microdialysis. Partial agonists may weakly activate the postsynaptic receptor or antagonize the actions of 5-HT_1A agonists such as 8-OH-DPAT, because little receptor reserve exists at these sites. However, partial agonists, such as buspirone, ipsapirone, and BMY 7378 may still behave as agonists at the autoreceptor due to the large receptor reserve associated with these cell bodies (Schechter et al., 2002). WAY-101405 demonstrated an antagonist profile, because administration of 1 mg/kg resulted in no change in extracellular 5-HT levels in terminal projection areas of the serotonergic cell bodies, whereas both 1 and 0.1 mg/kg completely antagonized 8-OH-DPAT-induced decreases in hippocampal 5-HT, indicating that this compound, similar to WAY-100635 (Forster et al., 1995) and lecozotan (Schechter et al., 2005), behaves as a silent antagonist in vivo.

There is an accumulating body of evidence supporting the role of 5-HT_1A receptors in cognitive function (Schechter et al., 2002). In the present studies, oral administration of WAY-101405 was shown to be effective in multiple animal models of learning and memory, with activity being observed at doses between 1 and 3 mg/kg. The novel object recognition task is based on the greater spontaneous behavior by rats to explore a novel object more than an object that they have explored previously (Ennaceur and Delacour, 1988). After a 48-h interval, or a 1-h interval for scopolamine-treated rats, the animals no longer discriminate between a known and unfamiliar object. Performance in this task is improved by a range of drugs, including 5-HT₆ receptor antagonists (Hirst et al., 2006), and it is impaired by scopolamine (Hirst et al., 2006). Acute administration of WAY-101405, given 2 h before training on day 1, significantly increased exploration of the novel object after both temporal and scopolamine-induced deficits, results similar to those reported previously with WAY-100635 (Pitsikas et al., 2003). We also investigated the effects of a combined treatment consisting of nonefficacious doses of both WAY-101405 and donepezil, and we demonstrated enhanced retention of recognition memory 48 h after training, a time where memory no longer can be measured in vehicle-treated animals. These results suggest that it may be possible, in patients, to combine a 5-HT_1A receptor antagonist with current therapies, such as cholinesterase inhibitors, and produce enhanced procognitive effects.

Contextual fear conditioning is an associative learning paradigm for studying aversive learning and memory. Previous work, in mice, has shown that 8-OH-DPAT interferes with acquisition, but not consolidation, in this model, an effect that was reversed by WAY-100635 (Stiedl et al., 2000). WAY-101405 reversed a scopolamine-induced deficit, in the present studies, in a rat contextual fear conditioning model. Although the precise mechanisms and processes of underlying memory formation in the brain are yet to be definitively described, it is generally recognized that the hippocampus plays a critical role in spatial memory (Martin and Clark, 2007), and our data suggest that hippocampal 5-HT_1A receptors play an important role in the limbic circuitry involved in contextual fear conditioning. This is further supported by the results from the Atlantis water maze studies, which clearly show that as the task of escaping from the water becomes harder, WAY-101405 significantly improves escape latency, demonstrating a positive effect on the encoding of spatial memory. Previous studies, directly investigating the role of the 5-HT_1A receptors in spatial memory in the water maze, have used scopolamine to impair performance, but they concluded that the sensorimotor effects of scopolamine confounded the results (Lüttgen et al., 2005). The majority of the reported studies with 5-HT_1A receptor antagonists, including the object recognition and contextual fear conditioning in the present study, have acutely administered the compounds. Given the potential use of this compound class in the treatment of cognitive dysfunction associated with psychiatric and neurological conditions, which is likely to require long-term administration, the effects of repeat dosing of WAY-101405 were an important factor to consider. The data from the water maze experiments demonstrate that daily treatment, for nine consecutive days, did not adversely affect the learning, on days 1 to 4, or the retention of the memory, on day 10, suggesting that there is no obvious tolerance effect.

In the present study, WAY-101405 was able to reverse a scopolamine-induced deficit, in both the novel object recognition and contextual fear conditioning paradigms, consistent with previous studies using other 5-HT_1A receptor antagonists (Schechter et al., 2002), suggesting that 5-HT_1A receptors may affect cognitive processes, at least in part, via the modulation of cholinergic neurotransmission as suggested by the localization of 5-HT_1A receptors. For example, 5-HT_1A receptors are localized on pyramidal neurons in the hippocampus (Chalmers and Watson, 1991); on cholinergic cell bodies in the medial septum and diagonal band of Broca, which innervate the hippocampus (Kia et al., 1996); and on cholinergic neurons in the nucleus basalis of Meynert, which projects to the frontal cortex (Van den Hooff and Galvan, 1992). 5-HT_1A autoreceptors may indirectly enhance corticolimbic acetylcholine release via relief of a tonic, inhibitory influence of serotonergic pathways upon cholinergic projections (Steckler and Sahgal, 1995). We measured acetylcholine levels in the hippocampus following WAY-101405 administration in the absence of acetylcholinesterase inhibitors, which may affect the drug action (Millan et al., 2004), and without the artificial stimulation of neurotransmitter release by K⁺ infusion (Schechter et al., 2005). Under these conditions, the WAY-101405-mediated release of acetylcholine in freely moving conscious rats was modest, but significant. These results are consistent with previously reported data on WAY-100635 (Millan et al., 2004), but we did observe a smaller overall effect, which could be due to the different strain of rat used or differences in the detection methods used. However, the increases that we measured were after a 10-mg/kg administration of WAY-101405, and although we did not test lower doses, it is possible that they would not have elicited a significant increase in acetylcholine levels, suggesting the cognitive effects, seen at doses of 1 or 3 mg/kg, may be a result of enhancing the release of other neurotransmitters, such as glutamate, which has been shown to be increased after WAY-101405 administration (Schechter et al., 2002).

The pharmacological effect of any drug is a function of its intrinsic efficacy and the extent to which it occupies the target, which is in turn dependent on affinity and concentration of the drug in the target tissue. We have used in vivo binding to determine receptor occupancy in the hippocampus and cerebral cortex of rats at efficacious doses of WAY-101405. 5-HT_1A receptor occupancy, at 2 h, the same time that cognitive assessments were made, was approximately 90% at doses of 1 and 3 mg/kg. We have recently demonstrated, in human patients using positron emission tomography imaging, that lecozotan can occupy up to 60% of 5-HT_1A receptors in elderly subjects and Alzheimer's disease patients (Raje et al., 2008), and clinical trials, measuring cognitive performance in similar patient populations are ongoing. The results of these studies will further increase our understanding of the role of 5-HT_1A receptors in human cognitive function.

In conclusion, we have demonstrated that WAY-101405 is a potent and selective, brain-penetrant, orally bioavailable 5-HT_1A receptor "silent" antagonist that is effective in preclinical memory paradigms at doses where approximately 90% of the postsynaptic 5-HT_1A receptors are occupied. These data further support the potential therapeutic utility of 5-HT_1A receptor antagonists in the treatment of cognitive dysfunction associated with psychiatric and neurological conditions.

	Acknowledgements

 
We thank Menelas N. Pangalos and Ronald L. Magolda for support of the 5-HT_1A receptor antagonist program.

	Footnotes

 
Parts of this work were presented previously at the following meetings: Schechter LE, Smith DL, Rosenzweig-Lipson S, Marquis K, Jones D, Nguyen HQ, Dawson LA, and Kelly MG (2000) Pharmacological characterization of an orally active 5-HT_1A receptor antagonist: WAY-405. The 30th Annual Meeting of the Society for Neuroscience; 2000 Nov 4–9; New Orleans, LA; and Hirst WD, Andree TH, Aschmies S, Childers W, Comery TA, Day M, Grauer SM, Hughes ZA, van der Lee H, Rosenzweig-Lipson S, et al. (2006) A novel silent 5-HT_1A receptor antagonist has efficacy in multiple rat cognition models. The 36th Annual Meeting of the Society for Neuroscience; 2006 Oct 14–18; Atlanta, GA. Society for Neuroscience, Washington DC.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.133082.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; WAY-100635, N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride; WAY-100135, N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropanamide; WAY-101405, (R)-N-(2-methyl-(4-indolyl-1-piperazinyl)ethyl)-N-(2-pyridinyl)-cyclohexane carboxamide; NAD-299, (R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate; MK-801, (–)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; h, human; r, rat; CHO, Chinese hamster ovary; HPLC, high-performance liquid chromatography; ANOVA, analysis of variance; LSD, least significant difference; BP, binding potential; BMY 7378, 8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione 2HCl; AUC, area under the curve; LY-426965, [(2S)-(+)-1-cyclohexyl-4-[4-(2-methoxyphenyl)-1-piperazinyl]2-methyl-2-phenyl-1-butanone monohydrochloride].

Address correspondence to: Dr. Warren D. Hirst, Discovery Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543. E-mail: hirstw{at}wyeth.com

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