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Correlating Efficacy in Rodent Cognition Models with In Vivo 5-HT1A Receptor

Occupancy by a Novel Antagonist, WAY-101405.

Warren D. Hirst, Terrance H. Andree, Suzan Aschmies, Wayne E. Childers, Thomas A. Comery,

Lee A. Dawson, Mark Day, Irene B. Feingold, Steven M. Grauer, Boyd L. Harrison, Zoë A.

Hughes, John Kao, Michael G. Kelly, Heidi van der Lee, Sharon Rosenzweig-Lipson, Annmarie

L. Saab, Deborah L. Smith, Kelly Sullivan, Stacey J. Sukoff Rizzo, Cesario Tio, Mei-Yi Zhang,

and Lee E. Schechter

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.L.,

S.R.-L., D.L.S., K.S., S.S.-R., L.E.S.) and Chemical & Screening Sciences (W.E.C., B.L.H.,

M.G.K., A.L.S., M-Y.Z.), Wyeth Research, Princeton, New Jersey; Drug Safety and Metabolism

(I.B.F., J.K. and C.T.), Wyeth Research, Collegeville, Pennsylvania.

JPET Fast Forward. Published on January 8, 2008 as DOI:10.1124/jpet.107.133082

Copyright 2008 by the American Society for Pharmacology and Experimental Therapeutics.

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Running title: Correlating Cognition with 5-HT1A Receptor Occupancy in Rats

Corresponding author:

Dr Warren D Hirst

Discovery Neuroscience

Wyeth Research

CN 8000

Princeton, NJ 08543

USA

Tel: +1 732 274 4001

Fax: +1 732 274 4020

e-mail: hirstw@wyeth.com

Number of text pages: 47

Number of tables: 2

Number of figures: 8

Number of references: 39

Number of words in Abstract: 245

Number of words in Introduction: 723

Number of words in Discussion: 1538

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List of non-standard abbreviations:

5-HT, 5-hydroxytryptamine; 8-OH-DPAT, 8-hydroxy-2-dipropylaminotetralin; ACh,

acetylcholine; aCSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; BMY 7378,

(8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione. 2HCl); BP,

binding potential, CHO, Chinese hamster ovary; HPLC, high-performance liquid

chromatography; LSD, least significant difference; LY426965, [(2S)-(+)-1-cyclohexyl-4-[4-(2-

methoxyphenyl)-1-piperazinyl]2-methyl- 2-phenyl-1-butanone monohydrochloride]; MK-801, (-

)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate; MS, mass

spectrometry; NAD-299, (R)-3-N,Ndicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-

5-carboxamide hydrogen (2R,3R)-tartrate monohydrate; NMDA, N-methyl-D-aspartate; NOR,

novel object recognition, PET, positron emission tomography; WAY-100135, N-tert-butyl-3-[4-

(2-methoxyphenyl)piperazin-1-yl]-2-phenylpropanamide; WAY-100635, (N-(2-(4-(2-

methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl)cyclohexanecarboxamide trihydrochloride;

WAY-101405, ((R)-N-(2-methyl-(4-indolyl-1-piperazinyl)ethyl)-N-(2-pyridinyl)-cyclohexane

carboxamide).

Recommended section assignment: Neuropharmacology

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Abstract

5-HT1A receptors play an important role in multiple cognitive processes and compelling evidence

suggests that 5-HT1A antagonists can reverse cognitive impairment. We have examined the

therapeutic potential of a potent (Ki = 1.1 nM), selective (> 100-fold), orally bioavailable, silent

5-HT1A receptor antagonist (KB = 1.3 nM), WAY-101405 ((R)-N-(2-methyl-(4-indolyl-1-

piperazinyl)ethyl)-N-(2-pyridinyl)-cyclohexane carboxamide). 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 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-HT1A receptors in the rat cortex with an ED50 of 0.1 mg/kg p.o..

Collectively these studies demonstrate that WAY-101405 is a potent and selective, brain

penetrant, orally bioavailable 5-HT1A receptor ‘silent’ antagonist that is effective in pre-clinical

memory paradigms at doses where approximately 90% of the post-synaptic 5-HT1A 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.

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Introduction

5-hydroxytryptamine (5-HT) exerts its diverse physiological and pharmacological effects

through actions on multiple receptor subtypes (reviewed by Hoyer et al., 2002), of which the 5-

HT1A receptor was the first described. The 5-HT1A receptor has been implicated in numerous

behavioral and physiological functions including learning and memory. This receptor is located

in brain regions associated with learning and memory, such as the hippocampus, frontal cortex,

and entorhinal cortex (Chalmers and Watson, 1991) and the cellular and sub-cellular localization

of the 5-HT1A receptors allows for direct and indirect modulation of multiple neurotransmitter

systems involved in cognitive processes (reviewed by Schechter et al., 2002).

The prototypical 5-HT1A 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 non-matching to position and 5-choice serial reaction task (reviewed by Schechter et al.,

2002 and reviewed by Myhrer, 2003). More recently, studies in human volunteers have shown

that oral administration of the 5-HT1A 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-HT1A receptors in the hippocampus as

assessed using [11C]WAY-100635 and positron emission tomography (Yasuno et al., 2003),

expanding on earlier studies which demonstrated changes in human 5-HT1A 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-HT1A receptor antagonist to be described (Fletcher et al.,

1993), but was later shown to have partial agonist activity at higher doses (Assie and Koek,

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1996). Subsequently, WAY-100635, an achiral analog of WAY-100135, was synthesized and

was shown to be the first selective and silent 5-HT1A receptor antagonist in a range of

pharmacological assays (Forster et al., 1995). Other 5-HT1A 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-HT1A 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-HT1A 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

rat whose nucleus basalis magnocellularis had been lesioned, but 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 (Luttgen et al., 2005) and passive

avoidance in mice (Madjid et al., 2006) and shown pro-cognitive effects.

Studies have also been described in non-human 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 NMDA 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).

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Although WAY-100635 has been used extensively in determining the consequences of 5-HT1A

receptor blockade it is not orally active and has a relatively short half-life that has precluded its

clinical development (Childers et al., 2005 and 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-HT1A

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-HT1A 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.

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Methods

Animals. Adult male Sprague-Dawley rats (225 –350g, 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 - 300g, Charles River Laboratories Inc.,

Wilmington, MA) 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 a.m.). Food and water were

available ad libitum, except where noted. All studies were previously approved by the

Institutional Animal Care and Use Committee and 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 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 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-

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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 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-HT1A Receptor Cloning and Preparation of Membranes. Stable Chinese hamster ovary cell

lines expressing the human 5-HT1A receptor subtype (h5-HT1A CHO cells) or rat 5-HT1A receptor

subtype (r5-HT1A CHO cells) were used throughout this study, the generation of these has been

previously described (Schechter et al., 2005). Cells were maintained in Dulbecco’s Modified

Eagle Medium supplemented with 10% fetal calf serum, nonessential amino acids, and

penicillin/streptomycin. Cells were grown to 95% to 100% confluency as a monolayer before

membranes were harvested for binding studies. Cells were gently scraped from the culture

plates, transferred to centrifuge tubes, and washed twice by centrifugation (2000 rpm for 10 min,

4°C) in buffer (50 mM Tris; pH 7.5). The resulting pellets were aliquoted and 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. 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-HT1A receptor binding studies were carried out in membranes

derived from h5-HT1A and r5-HT1A CHO cells in 50mM Tris-HCl, pH 7.4 in a 96-well microtiter

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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 non-specific binding was defined with 10 µM 5-HT. The

binding assays were initiated by the addition of 50 µl of the 5-HT1A cell membranes (0.05 mg

protein/sample) and were incubated at 25oC for 30 min. The reaction was terminated by vacuum

filtration through presoaked (0.5% polyethyleneimine) Unifilter GF/B filter plates (Perkin Elmer,

Boston, MA) using a Packard Filtermate 96 cell harvester. Microscint-20 scintillation cocktail

(Perkin Elmer, Boston, MA) was added and radioactivity was measured by liquid scintillation

spectrometry using a Packard TopCount liquid scintillation counter (Perkin Elmer, Boston, MA).

Protein concentrations were determined by the method of Bradford using bovine serum albumin

as the standard. Measurements were taken at 595 nm with a Pharmacia Biotech Ultraspec 3000

spectrophotometer (Pharmacia Biotech, Cambridge, England). 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). Ki values were then calculated from the IC50 value, as

previously described (Hirst et al., 2003), using KD 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 3 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-HT1A CHO cells,

grown in 96-well plates. Cells were pre-incubated at 37oC for 15 min in KREBS. Functional

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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, and incubated for an additional 10 min at 37oC. An apparent KB 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 above). Plates were incubated for 10 min at 37oC

and the assay was terminated by the addition of 0.5 M perchloric acid. Plates were stored at –

20oC 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 was

generated by a four parameter logistic equation using Prism (GraphPad Software Inc., San

Diego, CA) to determine the IC50 value. An apparent KB 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

intravenous bolus doses 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 diluted 1:1 with deionized water. In a separate study, brain tissue

was removed following an oral administration of WAY-101405 (0.3 mg/kg), washed free of

blood in sterile saline and homogenized in deionized water (50/50 v/v). Samples were extracted

using protein precipitation with acetonitrile containing an internal standard. The samples were

vortexed, centrifuged (3400 rpm for 5 min) and the supernatant was evaporated under an N2

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atmosphere at 37oC. The samples were reconstituted in acetonitrile/deionized water (50/50 v/v)

and analyzed for WAY-101405 concentration by reverse phase high-performance liquid

chromatography (HPLC)-tandem mass spectrometry (MS) 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 weighing 280 - 350 g at the time of surgery. Following 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, 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, USA) and two small stainless-steel screws (Plastics One). A subcutaneous cannula (s.c.)

was also implanted at this time between the animal’s shoulders. Following surgery, animals

were individually housed in Plexiglascages (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

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perfused with artificial cerebrospinal fluid (aCSF: 125 mM NaCl, 3 mM KCl, 0.75 mM MgSO4

and 1.2 mM CaCl2, pH 7.4) prior to insertion in the guide cannula. The microdialysis probe was

then inserted into the guide cannula and perfused with aCSF 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 prior to 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, following preinjection

baseline determination of 5-HT levels. Acetylcholine levels were determined in three control

samples, taken prior to 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 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, USA) and detected using an ANTEC electrochemical detector (ANTEC,

Netherlands) set at a potential of 0.65 V vs. a Ag/AgCl reference electrode. Mobile phase was

delivered by a Jasco PU980 HPLC pump (Jasco Ltd, Essex, U.K) at 0.5 ml/min and contained

0.15 M NaH2PO4 buffer at pH 4.6, 0.25 mM EDTA, 1.5 mM 1-octane sodium sulphonic acid and

10% methanol / 2% isopropanol. Data was acquired using the Atlas software package

(Labsystems, Gulph Mills, PA). The fmol perfusate values of 5-HT for the first four baseline

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samples were averaged and this value denoted as 100%. Subsequent sample values were

expressed as a percentage of this preinjection control value. All results were analyzed by 2 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 Inc., Berkeley, CA) for the PC.

Dialysate levels of acetylcholine were determined using LC/MS/MS as previously described

(Zhang et al., 2007). In brief, separation of acetylcholine was performed on a Supelco LC-SCX

5 uM HPLC column (2.1 x 150 mm) (Supelco, Bellefonte, PA, USA). Mobile phase A was

composed of 60%, 20%, 20% v/v of H2O, buffer (79.5 mM ammonium acetate, 63.5 mM

ammonium formate, pH 4.0), acetonitrile, respectively. Mobile phase B was composed of 20%,

80% v/v buffer (79.5 mM ammonium acetate, 63.5 mM ammonium formate, pH 4.0) and

acetonitrile, respectively. Eluates were detected using a Waters Micro mass spectrometer

coupled with Agilent HP 1100 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 acquired using Masslynx software (Micromass,

Beverly, MA, USA). Acetylcholine levels were expressed as a percentage of vehicle controls.

All results were analyzed by 2 way ANOVA with repeated measures followed by pairwise

comparisons using Bonferroni adjustment for multiple comparisons using SAS (v 1.03) within

Excel (Microsoft).

Novel Object Recognition. Male Long-Evans rats (180 - 200 g), housed individually, were

used in the novel object recognition (NOR) studies. This model is based on the greater

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spontaneous exploration of a novel object, compared to a familiar object, shown by rodents

(Ennaceur and Delacour, 1988). NOR training and testing was performed in a circular field

(diameter ~70cm, 30cm high) constructed out of plastic and containing soiled bedding. The field

was surrounded by black curtains to mask extra-field cues and was located in a dimly lit room

(~10 lux at the level of the area) in the presence of whitenoise (~65 dB). Animal performance

was tracked by video and 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 approximately 10 cm from the field’s edge. To avoid

possible olfactory cues multiple copies of the objects were used throughout the study and were

cleaned with a 30% ethanol solution between animals. Rats displayed no preference or aversion

to either of the objects and spent equivalent amount of time exploring objects if both were

presented simultaneously (data not shown). The visual recognition task was divided into 3

sessions - habituation, a sample trial and a choice trial. During habituation the animals were

placed into the field containing 2 identical yellow cubes (~10 x 10 x 10cm) and were allowed to

explore the field for 15 min. Following 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 <2cm of the object. Following the

sample trial rats were returned to their home cages for a 48 hour interval for the delay model or a

1 hour 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

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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

(v 1.03) within Excel). The amount of time exploring the novel and familiar objects across

treatment groups were 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. Significantly more time spent exploring the

novel object than the familiar one 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, St. Albans, VT). 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 stimulus and houselights and allowing exploration 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 co-terminated with the tone. This procedure was repeated, with the rats

remaining in the chamber prior to the second tone and footshock, the rats were removed from the

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chamber 30 s after the second footshock and returned to their homecage. Testing occurred

approximately 18-20 hours 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 analyzed using a one-way ANOVA

followed by Fisher’s LSD post hoc pairwise comparisons using SAS (v 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 prior to 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 upwards to provide lighting. The pool was filled with

water, to a depth of 0.3 m, that was made opaque with the addition of 150 ml of non-toxic

tempura paint (Michaels, The Arts & 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

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camera suspended centrally above the pool and all sessions were recorded on videotape. Data

were collected and analyzed on-line 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 prior to trial 1 on each of the 4 training days (n = 10 / treatment

group). Animals were also dosed, once daily, during the retention interval but did not receive

WAY-101405 during the retention tests on day 10.

Rats were pre-trained in the water maze with a visual cue task for 4 consecutive training trials on

one day (the starting points for each trial were randomized), each lasting a maximum of 60 s,

where the distal extra-maze cues were occluded by curtains around the circumference of the

pool. The escape platform was concealed from view 1 cm below the surface of the water and the

randomly allocated platform position was identified by a visual cue suspended above the escape

location. Escape latencies to the flagged platform were recorded, and rats were allowed to spend

30 s on the platform at the end of each training trial. Upon completion of visual cue training, rats

were ranked for average escape latency across these four trials, and they were evenly distributed

among the three treatment groups.

Following visual cue training all rats received 4 days of spatial training (4 trials per day) with the

curtains removed and the distal extra-maze 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 south-west 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). Utilizing the Atlantis platform (15 cm diameter, pneumatically

controlled submerged platform) rats were trained to dwell within a 35 cm diameter zone above

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the platform location for a predetermined period of 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 following 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 were expressed as a mean for each trials on each day, and are 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 to

4) followed by Dunnett’s post-hoc comparison using SAS (v 1.03) within Excel.

Five days after completion of the last acquisition session, rats were returned to the maze and

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. Percent 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 (v 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 prior to 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 prior to sacrifice (n = 4 / treatment

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group). Brains were immediately 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 hours of

shaking 30 µl of 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 transferred to a scintillation vial

containing 15 ml of Opti-Fluor (PerkinElmer) and the tritium content determined by liquid

scintillation spectrometry using a Canberra-Packard Tri-Carb 2200 liquid scintillation counter

(PerkinElmer). The 5-HT1A Binding Potential (BP) was determined for the frontal cortex and

hippocampus in each rat using the cerebellum, a brain region with relatively few 5-HT1A

receptors (Chalmers and Watson, 1991) as a reference tissue for the non-specific binding of

[3H]WAY-100635. Thus the BP is calculated as: (d.p.m. in frontal cortex – d.p.m. in

cerebellum) / d.p.m. in cerebellum (Wadenberg et al., 2000). The BP value for the vehicle

treated control group was pooled and the occupancy for each brain region in each rat was then

determined using the following formula: % Receptor Occupancy = 100 x ((5-HT1A control - 5-

HT1A individual) / 5-HT1A BP control) (Wadenberg et al., 2000).

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Results

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-HT1A receptors with Ki values of 1.13 ± 0.23 nM and

1.32 ± 0.22 nM, respectively (Table 1). WAY-101405 was selective for the 5-HT1A receptor

over other monoamine receptors that have previously been shown to have affinity for the aryl-

piperazine pharmacophore (Childers et al., 2005). A NovaScreen 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-HT1A receptor. At a

concentration of 100 nM WAY-101405 did not exhibit significant activity (> 50% inhibition) at

any of the 61 binding sites. Most notably, the compound was not active at various α-adrenergic

(1A, 1B, 2A, 2B), β-adrenergic (1, 2), adenosine (1, 2), dopamine (2, 3, 4), histamine (1, 2, 3),

muscarinic acetylcholine (1, 2, 3), or serotonin (1B, 1D, 2A, 2C, 3, 4, 5, 6, 7) receptors.

In functional assays with CHO cells expressing the human 5-HT1A 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 KB value of 1.27 ± 0.30 nM

(Table 1). WAY-101405 alone did not exhibit any agonist activity under any assay conditions.

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Phamacokinetics of WAY-101405. The pharmacokinetics of WAY-101405 was evaluated in

male Sprague-Dawley rats. Following a single intravenous 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

steady state distribution (Vss) of 3.8 l/kg, indicating moderate distribution into tissues, and an

apparent half-life of 0.8 h (Table 2). Following 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 2-4 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

when compared to 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. Pre-

treatment 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).

Evaluation of WAY-101405 in rodent models of cognition. The effect of a single dose of

WAY-101405 on temporal induced deficits in cognitive performance was investigated in a rat

novel object recognition model. Following a 48-hour delay, between training and testing trials,

vehicle-treated animals displayed no preferential exploration of the two objects suggesting a

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natural decay in their original memory for the original object (Fig. 3a). Administration of WAY-

101405 (0.3 - 10 mg/kg p.o.) resulted in a dose-dependent enhancement of the recognition

memory following the 48-hour delay in novel object recognition (Fig. 3a). Treatment with 1, 3

and 10 mg/kg of WAY-101405 (administered 2 hours prior to training on day 1) resulted in a

significantly (F (4,62) = 10.35, p < 0.001, post-hoc analysis (Fisher’s LSD) p < 0.05) greater

time spent exploring the novel than the familiar object demonstrating enhanced retention of the

previous learning experience. The 0.3 mg/kg treatment group did not display a preferential

exploration of the novel object. No significant effect of WAY-101405 on exploration during the

sample trial was observed (data not shown).

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 prior

to 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 as well as in rats treated with the groups that had received 1 and 3 mg/kg of 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, or 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, was not due to general changes in exploration of the objects (data not shown).

Collectively the data from the novel object recognition models demonstrated a minimal effective

dose in these paradigms of 1 mg/kg.

Using the recognition memory paradigm, we evaluated the pro-cognitive effects of WAY-

101405 in combination with a clinically utilized cognitive enhancer, donepezil. WAY-101405

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(0.3 mg/kg, p.o.) or donepezil (an acetylcholinesterase inhibitor, 0.5 mg/kg, i.p.), alone or in

combination, did not affect overall exploration time during the sample trial (Fig. 4a) (F (3,36) =

2.601, p = 0.0670). Consistent with previous results, 0.3 mg/kg of WAY-101405, administered

alone, was a non-efficacious dose in the 48-hour delay rat novel object recognition model.

Donepezil dose response experiments (data not shown) revealed that a non-efficacious dose of

this compound was 0.5 mg/kg i.p.. A combined treatment consisting of non-efficacious doses of

WAY-101405 and donepezil significantly enhanced retention of recognition memory 48 h after

training, a time where memory no longer can be measured in vehicle-treated animals (Fig. 4b) (F

(3,36) = 1.75, p = 0.1733, post-hoc analysis (Fisher’s LSD) p < 0.05).

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 prior to training on day 1, caused a robust

deficit in the amount of time the rats spent freezing when re-introduced 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 prior to 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).

WAY-101405 (0.3 and 3 mg/kg p.o.) was evaluated in a hippocampal dependent spatial memory

task, the Atlantis water maze (Fig. 6a). In the Atlantis water maze, animals do not have ready

access to the escape platform, but must spend a predetermined amount of “dwell time” in a

“trigger zone”, which encompasses the spot in which a pneumatically activated platform rises

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from the bottom of the water tank. Increasing dwell times over training sessions is a way to non-

pharmacologically increase the difficulty of the task, thus providing for an expanded window to

see improvements with test compounds. On the first training day, vehicle and drug treated rats

learn the location the escape platform, using the extra-maze cues, from trial 1 to trial 4, as

reflected by the progressive decrease in the mean escape latency. As the task becomes

progressively more demanding on the subsequent days, when the dwell time over the trigger

zone increases to 2 s and then 3 s, the vehicle and 0.3 mg/kg groups find it harder to locate the

platform with the mean escape latencies only improving moderately across the 4 trials. In

contrast, the rats which had received 3 mg/kg p.o. of WAY-101405 demonstrated an

improvement by trial 4, or earlier, when compared to vehicle treated animals, demonstrating a

positive effect on the encoding of spatial memory (Fig. 6b). Statistical analysis of the escape

latencies (treatment by trial) for each day gave the following values; day 1: F (6,81) = 1.51, p =

0.1858; day 2: F (6,81) = 0.55, p = 0.7702; day 3: F (6,81) = 1.20, p = 0.3151; day 4: F (6,81) =

2.76, p = 0.0173. Post-hoc analysis (Dunnett’s) showed that the rats which had received 3 mg/kg

p.o. of WAY-101405 demonstrated a significant improvement in trials 2, 3 and 4 (p < 0.001) on

day 4, when compared to vehicle treated animals.

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).

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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).

In vivo receptor occupancy. The high affinity, selective and brain penetrant 5-HT1A antagonist,

WAY-100635, has been radiolabeled and used for in vivo binding studies in rats (Hume et al.,

1994) and humans, in the form of PET (Pike et al., 1995). The tritiated form of WAY-100635

was used in the present studies to measure in vivo receptor occupancy by orally administered

WAY-101405. 5-HT1A receptor occupancy by WAY-101405, in both the frontal cortex and

hippocampus, increased in a dose dependent manner with > 90% occupancy observed at the

highest doses (1 and 3 mg/kg). In the vehicle treated animals the mean d.p.m. / mg protein in

each brain region were 3304 ± 105, 2716 ± 151 and 493 ± 26 for hippocampus, frontal cortex

and cerebellum, respectively. The cerebellum was used as a reference tissue for the non-specific

binding of [3H]WAY-100635, which, under the present conditions, accounted for 15 – 18% of

the total binding.

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Discussion

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-HT1A receptor in radioligand binding studies and 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 2-4 h. Previously reported pharmacodynamic effects, in fixed ratio operant response

studies in rats (Schechter et al., 2000 and unpublished observations), showed a good correlation

with the pharmacokinetic data reported in the present study and these combined results

determined the 2 h pre-treatment time that was used in the cognitive assays and receptor

occupancy studies.

The estimation of the intrinsic activity of 5-HT1A receptor ligands is pivotal in characterizing the

pharmacological and potential therapeutic utility of a specific compound of this class. Central 5-

HT1A receptors exist both in presynaptic (somatodendritic autoreceptor) and postsynaptic

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locations within the central nervous system, and 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-HT1A

autoreceptors localized on the cell bodies in the mid-brain have been measured by

electrophysiological techniques and neurochemical techniques such as in vivo microdialysis.

Partial agonists may weakly activate the post-synaptic receptor or antagonize the actions of 5-

HT1A agonists such as 8-OHDPAT since 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

(Fletcher et al., 1996) 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-HT1A 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 one that they have

previously explored (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

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object. Performance in this task is improved by a range of drugs, including 5-HT6 receptor

antagonists (Hirst et al., 2006), and is impaired by scopolamine (Hirst et al., 2006). Acute

administration of WAY-101405, given 2 h prior to training on day 1, significantly increased

exploration of the novel object following both temporal and scopolamine induced deficits, results

similar to those previously reported with WAY-100635 (Pitsikas et al., 2003). We also

investigated the effects of a combined treatment consisting of non-efficacious doses of both

WAY-101405 and donepezil and 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-HT1A receptor

antagonist with current therapies, such as cholinesterase inhibitors, and produce enhanced pro-

cognitive 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-HT1A 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

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role of the 5-HT1A receptors in spatial memory in the water maze, have used scopolamine to

impair performance but concluded that the sensorimotor effects of scopolamine confounded the

results (Luttgen et al., 2005). The majority of the reported studies with 5-HT1A 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 demonstrates

that daily treatment, for 9 consecutive days, did not adversely affect the learning, on days 1-4, or

the retention of the memory, on day 10, suggesting that there is no obvious tolerance effect.

In the present studies 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-HT1A receptor antagonists (Schechter et al., 2002) suggesting that

5-HT1A receptors may affect cognitive processes, at least in part, via the modulation of

cholinergic neurotransmission as suggested by the localization of 5-HT1A receptors. For

example, 5-HT1A 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-

HT1A 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

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(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

following a 10 mg/kg administration of WAY-101405 and although we didn’t test lower doses, it

is likely 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 following

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-HT1A 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 PET

imaging, that lecozotan can occupy up to 60% of 5-HT1A 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-HT1A receptors in human cognitive function.

In conclusion, we have demonstrated that WAY-101405 is a potent and selective, brain

penetrant, orally bioavailable 5-HT1A receptor ‘silent’ antagonist that is effective in pre-clinical

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memory paradigms at doses where approximately 90% of the post-synaptic 5-HT1A receptors are

occupied. These data further support the potential therapeutic utility of 5-HT1A receptor

antagonists in the treatment of cognitive dysfunction associated with psychiatric and

neurological conditions.

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Acknowledgements

We thank Menelas N. Pangalos and Ronald L. Magolda for their support of the 5-HT1A receptor

antagonist program.

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Footnotes

Portions of this work were presented previously at the following meetings:

L.E. Schechter, D.L. Smith, S. Rosenzweig-Lipson, K. Marquis, D. Jones, H.Q. Nguyen, L.A.

Dawson and M.G. Kelly (2000) Pharmacological characterization of an orally active 5-HT1A

receptor antagonist: WAY-405. The 30th Annual Meeting of the Society for Neuroscience, New

Orleans.

W.D. Hirst, T.H. Andree, S. Aschmies, W. Childers, T.A. Comery, M. Day, S.M. Grauer, Z.A.

Hughes, H. van der Lee, S. Rosenzweig-Lipson, D.L. Smith, K. Sullivan, S.J. Sukoff Rizzo,

M.Y. Zhang and L.E. Schechter (2006) A novel silent 5-HT1A receptor antagonist has efficacy in

multiple rat cognition models. The 36th Annual Meeting of the Society for Neuroscience,

Atlanta.

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Legends for Figures

Fig. 1. Chemical structure of WAY-101405

Fig. 2. WAY-101405 blocks an 8-OH-DPAT mediated decrease on 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 pre-injection baseline values from 14 rats (vehicle), 6 rats (WAY-101405), 7 rats

(WAY-101405 plus 8-OH-DPAT), 8 rats (8-OH-DPAT) (*, p < 0.05). Baseline 5-HT levels

were 42.8 ± 2.7 fmol/20 µl.

Fig. 3. Effects of WAY-101405 (0.3 to 10 mg/kg p.o., administered 2 h prior to 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 prior to training on day 1) (b) in a rat novel object recognition paradigm.

Mean time (s) ± S.E.M. spent exploring novel and familiar objects is shown from 10 rats per

treatment group (*, p < 0.05 versus familiar).

Fig. 4. 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 (a). Effects of donepezil (0.5 mg/kg

i.p., administered 30 min prior to training on day 1) and WAY-101405 (0.3 mg/kg p.o.,

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administered 2 h prior to training on day 1) alone and in combination on a temporal induced

deficit (48 h) in a rat novel object recognition paradigm (b). Mean time (s) ± S.E.M. spent

exploring novel and familiar objects is shown from 10 rats per treatment group (*, p < 0.05

versus familiar).

Fig. 5. Effects of WAY-101405 (0.3 to 3 mg/kg p.o., administered 2 h prior to 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 percent time spent freezing of 8 rats per treatment

group (*, p < 0.05 versus vehicle/vehicle group; #, p < 0.05 versus scopolamine/vehicle group).

Fig. 6. Effects of WAY-101405 in a water maze test. Rats were pre-trained using a visual cue in

4 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 prior to trial 1 on each of the 4 training days. Animals were

also dosed, once daily, during the retention interval but did not receive WAY-101405 during the

retention tests on day 10 (a). Acquisition curves across 4 consecutive training trials on days 1-4

following 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 (s) of 10 rats per treatment group (***, p < 0.001 versus

vehicle group) (b). Retention tests measured percent 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 %

time in target zone of 10 rats per treatment group (***, p < 0.001 versus vehicle group) (c).

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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 7 rats per vehicle group and 4 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).

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. 7.5 µCi of [3H]WAY-100635 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 non-specific binding from 4 rats per

treatment group.

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Table 1. In vitro pharmacological profile of WAY-101405. Data are expressed as mean ±

S.E.M.

Parameter Assay system Value

Binding

affinity

Recombinant human 5-HT1A receptors:

[3H]8-OH-DPAT binding

Ki = 1.13 ± 0.23 nM

Recombinant rat 5-HT1A receptors:

[3H]8-OH-DPAT binding

Ki = 1.32 ± 0.22 nM

Functional

potency

Recombinant human 5-HT1A receptors:

cAMP accumulation assay

KB = 1.27± 0.30 nM

Selectivity 61 neurotransmitter receptors, transporters,

ion channels and enzymes sites – NovaScreen

panel

> 100-fold selective

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Table 2. Pharmacokinetic properties of WAY-101405.

Route of administration and dose Parameter Value

Intravenous (i.v.) 1 mg/kg CLP (l/h/kg) 3.9

VSS (l/kg) 3.8

AUC0-inf (ng.h/ml) 260

Apparent t1/2 (h) 0.8

Oral (p.o.) 10 mg/kg Cmax (ng/ml) 1640

Tmax (h) 0.25

t1/2 (h) 3.1

AUC0-inf (ng.h/ml) 7160

% F 22

Plasma concentration at 2 h (ng/ml) 1020

Brain concentration at 2 h (ng/g) 4015

Mean Brain:Plasma (AUC) 5:1

N

HN

NN

N

O

Fig 1

vehicle

0.3 mg/kg 8-OHDPAT

1 mg/kg WAY-101405

0.01 mg/kg WAY-101405 & 8-OH-DPAT

0.1 mg/kg WAY-101405 & 8-OH-DPAT

1 mg/kg WAY-101405 & 8-OH-DPAT

0

50

100

150

0 50 100 150 200 250 300 350

Time (min)

**

**

*

***

*****

***

*

*

WAY-101405 8-OH-DPAT

5-H

T in

th

e H

ipp

oca

mp

us

(% o

f B

asel

ine)

Fig 2

10203040

50

VehicleVehicle

Scop.Vehicle

Scop.0.3 mg/kg

Novel

Familiar

*

Scop.1 mg/kg

Scop.3 mg/kg

WAY-101405 (p.o.) 2 h pre-treatment

**

Co

nta

ct T

ime

(s)

b

1020304050

Vehicle 1 mg/kg 3 mg/kg 10 mg/kg

WAY-101405 (p.o.) 2 h pre-treatment

0.3 mg/kg

* * *

Co

nta

ct T

ime

(s)

a

Fig 3

0

20

40

60

80

Vehicle Aricept0.5 mg/kg

WAY-1014050.3 mg/kg

WAY-101405+ Aricept

Co

nta

ct T

ime

(s)

a

b

Novel

Familiar

0

10

20

30

40

50

Co

nta

ct T

ime

(s)

Vehicle Aricept0.5 mg/kg

WAY-1014050.3 mg/kg

WAY-101405+ Aricept

*

Fig 4

10

20

30

40

50

60%

Tim

e S

pen

t F

reez

ing

#

VehicleVehicle

Scop.Vehicle

Scop.0.3 mg/kg

Scop.1 mg/kg

Scop.3 mg/kg

WAY-101405 (p.o.) 2 h pre-treatment

**

*

Fig 5

a

b

c

Training (Learning)

Retention Tests (Memory)

Days 1 - 4 Days 5 - 9 Day 10

Retention Interval

Vehicle, 0.3 or 3 mg/kg p.o WAY-101405

Pre-TrainingVisual Cue

Training (Learning)

Retention Tests (Memory)

Days 1 - 4 Days 5 - 9 Day 10

Retention Interval

Vehicle, 0.3 or 3 mg/kg p.o WAY-101405

Pre-TrainingVisual Cue

Vehicle 0.3 mg/kg 3 mg/kg0

10

20

30

40

50

***

% T

ime

in T

arg

et Z

one

Vehicle 0.3 mg/kg 3 mg/kg0

10

20

30

40

50

***

% T

ime

in T

arg

et Z

one

Fig 6

T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T4 T1 T2 T3 T40

20

40

60

80

100

120

Day 1 Day 2 Day 3 Day 4

*********

Esc

ape

Lat

ency

(s)

Vehicle0.3 mg/kg3 mg/kg

-10

0

10

20

30

40

50

-80 -40 0 40 80 120 160 200Time (min)

AC

h(%

ch

ang

e fr

om

veh

icle

)

VehicleWAY-101405 (10 mg/kg p.o.)

*

Fig 7

10

20

30

40

50

60

70

80

90

100

0.1 mg/kg 0.3 mg/kg 1 mg/kg 3 mg/kg

WAY-101405 (p.o.) – 2h pre-treatment

% R

ecep

tor

Occ

up

ancy

Frontal Cortex

Hippocampus

Fig 8