JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 JPET FastThis articleForward. has not Published been copyedited on and October formatted. 14,The final2011 version as DOI:10.1124/jpet.111.186585 may differ from this version.

Title Page

CEP-26401 (Irdabisant), a potent and selective H3 /

inverse agonist with cognition-enhancing and wake promoting activities

Downloaded from

Rita Raddatz, Robert L. Hudkins, Joanne R. Mathiasen*, John A. Gruner*, Dorothy G.

Flood*, Lisa D. Aimone, Siyuan Le*, Hervé Schaffhauser*, Maciej Gasior*, Donna jpet.aspetjournals.org

Bozyczko-Coyne, Michael J. Marino*, Mark A. Ator, Edward R. Bacon, John P.

Mallamo and Michael Williams* at ASPET Journals on September 24, 2021

Discovery Research

Cephalon, Inc.

145 Brandywine Parkway

West Chester, PA 19380

USA

Copyright 2011 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 2

Running Title CEP-26401

Corresponding author: Rita Raddatz

Cephalon, Inc.

145 Brandywine Parkway

West Chester, PA 19380 USA

PHONE : (610) 738-6728 FAX: (610) 738-6305 Downloaded from [email protected]

Text Pages: 48

Tables: 6 jpet.aspetjournals.org

Figures: 7

References: 40 at ASPET Journals on September 24, 2021 Abstract (# of words): 240

Introduction (# of words): 490

Discussion (# of words): 1500

Abbreviations: H3R, ; CEP-26401, 6-{4-[3-((R)-2-methyl- pyrrolidin-1-yl)-propoxy]-phenyl}-2H-pyridazin-3-one HCl; ABT-239, 4-(2-{2-[(2R)-2- methyl-1-pyrrolidinyl]ethyl}-1-benzofuran-5-yl)benzonitrile (L) tartrate; RAMH, R-α- methylhistamine; GTPγS, guanosine 5`-(γ-thio)triphosphate; PPI, prepulse inhibition;

[3H]NAMH, [3H]N-α-methylhistamine; RID, ratio of investigation duration; DTT, dithiothreitol; EEG, electroencephalographic; EMG, electromyographic; SWS, slow wave sleep; REMS, rapid eye movement sleep.

Recommended section: Neuropharmacology JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 3

Abstract

CEP-26401 (Irdabisant) is a novel, potent histamine H3 receptor (H3R) antagonist/ inverse agonist with drug-like properties. High affinity of CEP-26401 for H3R was demonstrated in radioligand binding displacement assays in rat brain membranes (Ki =

2.7 ± 0.3 nM) and in recombinant rat and human H3R-expressing systems (Ki = 7.2 ± 0.4

nM, Ki = 2.0 ± 1.0 nM, respectively). CEP-26401 displayed potent antagonist and Downloaded from inverse agonist activities in [35S]GTPγS binding assays. Following oral dosing of CEP-

26401, occupancy of H3R was estimated by inhibition of ex vivo binding in rat cortical jpet.aspetjournals.org slices (OCC50 = 0.1 ± 0.003 mg/kg) and antagonism of the H3R agonist, R-α- methylhistamine (RAMH), -induced drinking response in the rat dipsogenia model was demonstrated in a similar dose range (ED50 = 0.06 mg/kg). CEP-26401 improved at ASPET Journals on September 24, 2021 performance in the rat social recognition model of short-term memory at doses of 0.01-

0.1 mg/kg p.o. and was wake-promoting at 3-30 mg/kg p.o. In DBA/2NCrl mice, CEP-

26401 at 10 and 30 mg/kg i.p. increased prepulse inhibition (PPI), while the antipsychotic was effective at 0.3 and 1 mg/kg i.p. Co-administration of CEP-26401 and risperidone at sub-efficacious doses (3 mg/kg i.p. and 0.1 mg/kg i.p., respectively) increased PPI. These results demonstrate potent behavioral effects of CEP-26401 in rodent models and suggest that this novel H3R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. CEP-26401 may also have therapeutic utility in treating schizophrenia or as adjunctive therapy to approved antipsychotics.

JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 4

Introduction

The CNS system is involved in regulation of sleep-wake, attention and metabolic homeostasis via activation of a family of G-protein-coupled receptors (GPCRs) including H1, H2, H3 and H4 histamine receptors (Leurs et al., 2011). Of these, the H3 (H3R) was identified as a presynaptic autoreceptor modulating neuronal histamine release (Arrang et al., 1983). Subsequent studies demonstrated that

H3R also functions as a heteroreceptor to modulate the release of other neurotransmitters Downloaded from associated with cognitive function and the regulation of sleep-wake, including acetylcholine, norepinephrine, dopamine, and serotonin (Esbenshade et al., 2008). jpet.aspetjournals.org

The discovery of the H3R antagonists and ciproxifan provided the pharmacological tools required for the exploration of the role of the H3R in CNS function. While active in multiple preclinical models, these early H3R antagonists were at ASPET Journals on September 24, 2021 limited as putative drug candidates due to poor target selectivity, lack of metabolic stability, potent hERG interactions, cytochrome P450 inhibition and poor brain penetration. Differences in pharmacology between the rodent and human H3Rs were also identified that hindered early drug discovery (Ireland-Denny et al., 2001). Considerable efforts in academia and industry resulted in the discovery of H3R antagonists / inverse agonists that have overcome many of these issues and provided tools to enhance understanding of H3R functions, with several compounds in clinical trials (Raddatz et al.,

2010; Brioni et al., 2011; Berlin et al., 2011).

The histaminergic system in the brain is active during wakefulness, and inhibitors of post-synaptic H1 histamine receptors are well established sedatives. The ability of H3R JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 5 antagonists to increase activity in the brain histaminergic system and enhance vigilance and wakefulness has been demonstrated in multiple species (Parmentier et al., 2007). In addition to the effects of H3R antagonists on wake promotion in preclinical models, they are effective in decreasing episodes of cataplexy in a genetically narcoleptic Doberman

Pinscher model (Bonaventure et al., 2007), suggesting the potential to treat multiple symptoms of . A number of H3R antagonists have also demonstrated efficacy

across a broad range of preclinical models of cognitive function, including short- and Downloaded from long-term memory, working memory, spatial memory and sensorimotor gating

(Esbenshade et al., 2008). H3R antagonists are currently being targeted for clinical use in jpet.aspetjournals.org the treatment of attention-deficit hyperactivity disorder (ADHD), Alzheimer’s disease

(AD), schizophrenia and other disorders associated with cognitive impairment, as well as in the treatment of narcolepsy and sleep-wake disorders (Raddatz et al., 2010; Brioni et at ASPET Journals on September 24, 2021 al., 2011; Berlin et al., 2011; Leurs et al., 2011).

In the present report, the pharmacological properties and behavioral effects of the novel

H3R antagonist/ inverse agonist, CEP-26401 (6-{4-[3-((R)-2-methyl-pyrrolidin-1-yl)- propoxy]-phenyl}-2H-pyridazin-3-one; irdabisant; Hudkins et al., 2011) (Figure 1), are described. This selective, high affinity H3R antagonist potently enhanced short-term memory, improved PPI and demonstrated robust wake promotion in preclinical models, confirming the potential utility of H3R antagonists for the treatment of cognitive/ attentional disorders as well as sleep disorders. JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 6

Methods

Chemicals

CEP-26401 and ABT-239 (Figure 1) were synthesized at Cephalon, Inc. (West Chester,

PA) by methods previously described (Hudkins et al., 2011; Cowart et al., 2004). R-α- methylhistamine (RAMH), , hydrochloride, guanosine 5`-(γ- thio)triphosphate (GTPγS), thioperamide and ciproxifan were purchased from Sigma-

Aldrich (St. Louis, MO). and dihydrobromide were purchased from Downloaded from

Tocris (Ellisville, MO) and risperidone was purchased from Interchem (Paramus, NJ).

[3H]N-α-methylhistamine ([3H]NAMH, 45-90 Ci/mmol), [3H]tiotidine (86 Ci/mmole), jpet.aspetjournals.org and [35S]GTPγS (1250 Ci/mmol) were purchased from PerkinElmer (Boston, MA).

[3H]Pyrilamine (32 Ci/mmol) and [2,5-3H]histamine dihydrochloride ([3H]histamine, 30-

60 Ci/mmol) were purchased from GE Healthcare (Piscataway, NJ). at ASPET Journals on September 24, 2021

Receptor cloning and cell growth

The cDNA encoding the 445-amino-acid splice variant of the rat histamine H3R (rH3R)

(b.p. 338-1675 of GenBank accession no. NM_053506) was amplified from rat cDNA prepared from RNA isolated from thalamus, hypothalamus, striatum and prefrontal cortex. The sequence-verified cDNA was inserted into the pIRESneo3 vector (Clontech

Laboratories, Inc., Palo Alto, CA) and the resulting construct was transfected into

Chinese hamster ovary (CHO-A3) cells (cell line expressing mitochondrially targeted apoaequorin and human Gα16 via transfection of the pCAeqG15 plasmid (Euroscreen

S.A., Gosselies, Belgium) using Lipofectamine™ Transfection Reagent (Invitrogen

Corp., Carlsbad, CA). Cells having incorporated the plasmid DNA were selected by the addition of 1.5 mg/ml G418 to the growth media 48 h after transfection, and a clonal line JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 7

was established following limiting dilution. The rH3R cell line was grown in Ham’s F12 with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, 100 µg/ml streptomycin, 250

µg/ml Zeocin™ and 400 µg/ml G418 at 37ºC with 5% CO2. CHO-K1 cells expressing the full length human H3R (hH3-C1 cell line) were purchased from Euroscreen S.A. and were grown in Ham’s F12 with 10% FBS, 100 IU/ml penicillin, 100 µg/ml streptomycin and 400 µg/ml G418 at 37ºC with 5% CO2.

The cDNA encoding the full-length 391-amino-acid human (b. Downloaded from p. 101-1273 of GenBank accession no. NM 021624) was PCR-amplified from human bone marrow and spleen cDNAs (Clontech Laboratories, Inc., Mountain View, CA). The jpet.aspetjournals.org sequence-verified cDNA was inserted into the bicistronic mammalian expression vector, pIRESneo3 (Clontech Laboratories, Inc.) and was transfected into CHO-K1 cells

(ATCC® #CCL-61™, Manassas, VA) using Lipofectamine™ Transfection Reagent. at ASPET Journals on September 24, 2021

Cells having incorporated the plasmid DNA were selected for by the addition of 1.5 mg/ml G418 to the media 48 h after transfection. After selection for 2 weeks, clones were generated by limiting dilution in 96-well plates. Cells were grown in Ham’s F12 with 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml G418 at

37ºC with 5% CO2.

Membrane preparations

Rat corical membranes were prepared as previously described (Le et al., 2009) and stored at -80ºC until use. Membranes from cells expressing rH3R, hH3R or hH4R were prepared by resuspending frozen pellets of cells in membrane buffer (5 mM Tris-HCl (pH 7.5), 5 mM EDTA and protease inhibitors (complete EDTA-free protease inhibitor tablets,

Roche Applied Science, Indianapolis, IN)), homogenizing the cell pellets, removing the JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 8 pellet formed following centrifugation at 1000g for 10 min at 4ºC and centrifuging the supernatant at 40,000g for 30 min at 4ºC. The resulting membrane pellets were washed by resuspension in membrane buffer (50 mM Tris-HCl (pH 7.5), 0.6 mM EDTA, 5 mM

MgCl2 and protease inhibitors) and collection by centrifugation as described above. Final membrane pellets were resuspended in membrane buffer containing 250 mM sucrose and frozen at -80 ºC until use. Protein concentration was determined using the Coomassie

Plus kit (Pierce Biotechnology, Rockford, IL). Downloaded from

Radioligand binding assays

Rat cortical membranes were thawed on ice, washed three times with 30 ml TE buffer jpet.aspetjournals.org (50 mM Tris (pH 7.4) with 5 mM EDTA) using a polytron and centrifuged at 39,000g for

10 min at 4ºC. Radioligand binding to the H3R was performed by modification of a previously described method (Kilpatrick and Michel, 1991). Washed membranes (200 at ASPET Journals on September 24, 2021

μg protein) were incubated with 0.2 nM of [3H]NAMH with or without various concentrations of test compound at 30ºC for 40 min in a total volume of 500 μl. The reaction was stopped by rapid filtration through presoaked (0.3% polyethyleneimine) glass fiber filters (GF/B, FPLR-124, Brandel, Gaithersburg, MD) using a Brandel Cell

Harvester (Gaithersburg, MD). The filters were washed five times with 1 ml of cold TE buffer, mixed with 5 ml of liquid scintillation cocktail (Ultima Gold, PerkinElmer Life

Science, Shelton, CT), and measured using a liquid scintillation counter (Tri-Carb

3100TR, PerkinElmer Life Science). Nonspecific binding was defined in the presence of

10 μM of thioperamide.

Recombinant rH3R- or hH3R-expressing membranes were diluted to 0.5-1 mg protein/ml in 50 mM Tris-HCl (pH 7.5), 0.1% bovine serum albumin (BSA), 5 mM MgCl2 buffer JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 9 and incubated for 4 h at 24°C with 0.8-2 nM [3H]NAMH, vehicle or test compound, and

0.5 mg/well FlashBlue scintillation beads (SPA beads; PerkinElmer) in a final volume of

80 µl/well in a 96-well plate. Nonspecific binding was determined in the presence of 1

3 µM clobenpropit. Binding of [ H]NAMH to the hH3R membranes was saturable with

Bmax = 1.9 ± 0.7 pmole/mg protein and Kd = 1.2 ± 0.2 nM. The rat H3R cell membranes

3 expressed 1.1 ± 0.3 pmole/mg protein binding sites for [ H]NAMH with Kd = 0.8 ± 0.2

nM. The saturation binding isotherms were best fit to a single site. Membranes Downloaded from expressing hH1 receptors (10 µg protein; PerkinElmer; ES-390-M400UA) were incubated

3 with 1.5 nM [ H]pyrilamine (Kd = 1.5 nM) and 0.5 mg FlashBlue scintillation beads in a jpet.aspetjournals.org final volume of 80 µl buffer (50 mM Tris (pH 7.4), 5 mM MgCl2, 0.2 % w/v BSA) with or without test compounds for 3 h at room temperature. Nonspecific binding was determined in the presence of 5 μM triprolidine. Membranes expressing hH2 receptors at ASPET Journals on September 24, 2021

(2.5 µg protein; PerkinElmer; ES-391-M400UA) were incubated with 5 nM [3H]tiotidine

(Kd = 2.7 nM) and 0.25 mg scintillation beads in a final volume of 80 µl buffer (50 mM

HEPES (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 1 mM CaCl2 and 0.2% w/v BSA) with or without test compounds for 4 h at room temperature. Nonspecific binding was determined in the presence of 32 μM ranitidine. Bound radioligand was determined by counting on a Wallac MicroBeta® TriLux scintillation counter (PerkinElmer) for all the scintillation bead assays.

Membranes (10 μg protein) expressing hH4 receptors were incubated with 15 nM

3 [ H]histamine (Kd = 5.8 nM), with or without test compounds in a final volume of 200 µl buffer (50 mM Tris HCl (pH 7.5), 5 mM EDTA, 10 μg/ml saponin) for 1 h at room temperature and then filtered through a 96-well filter plate presoaked in 0.3 % w/v JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 10 polyethylenimine (Sigma-Aldrich) in wash buffer (cold 50 mM Tris HCl, pH 7.5) using a

Brandel Cell Harvester. Filter plates were rapidly washed four times, allowed to dry and

Betaplate scintillant (50 μl per well; Perkin Elmer) was added. Bound radioactivity was counted in a Wallac MicroBeta® TriLux scintillation counter. Nonspecific binding was determined in the presence of 20 μM imetit.

In vitro functional characterization by [35S]GTPγS binding assay

Antagonist potency was determined by measuring inhibition of RAMH-induced Downloaded from

35 [ S]GTPγS binding in recombinant hH3R and rH3R membranes. Frozen aliquots of membranes containing recombinant hH3 or rH3 were thawed and diluted in assay buffer jpet.aspetjournals.org

(200 mM NaCl, 20 mM MgCl2, 1 mM EDTA and 1 mM dithiothreitol (DTT) with 30

μg/ml saponin in 20 mM HEPES, pH 7.4) to a final concentration of 0.5 mg protein per ml. Membranes were pre-mixed with 0.5 mg SPA beads per 10 µg membrane protein at ASPET Journals on September 24, 2021 and 20 µM GDP in a 96-well plate. Test compounds or vehicle plus [35S]GTPγS (final concentration = 0.2 nM) were added. RAMH (100 nM), a concentration producing approximately 80% of maximum RAMH-induced signal in these assays, was added and the plates were incubated for 45 min at room temperature. Following centrifugation at

22ºC at 250g for 5 min, bound radioactivity was measured using a Wallac MicroBeta®

TriLux scintillation counter. Isolated rat cortical membranes (50 μg) were incubated with

0.1 nM [35S]GTPγS, 400 μM GDP and 100-220 nM RAMH, with or without test compounds, in assay buffer (50 mM Tris (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 1 mM

EDTA and 0.16 µg/ml DTT) for 60 min at 30ºC. The reaction was stopped by rapid filtration onto GF/C plates (PerkinElmer) using a Brandel Cell Harvester and washed five times with 500 μl wash buffer (20 mM HEPES, 100 mM NaCl, 20 mM MgCl2, 1 mM JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 11

EDTA and 0.16 μg/ml DTT). The plates were dried, mixed with 50 μl scintillant (Beta

Scin, PerkinElmer) and counted in a Wallac MicroBeta® TriLux scintillation counter.

Inverse agonist potency was determined by measuring inhibition of basal [35S]GTPγS binding in recombinant hH3R and rH3R membranes. Membranes were diluted in buffer

(100 mM NaCl, 5 mM MgCl2, 1 mM EDTA and 1 mM DTT with 30 μg/ml saponin in 20 mM HEPES, pH 7.4) to provide 10 µg protein per 20 µL. Diluted membranes and SPA

beads (0.5 mg/well) were pre-mixed with GDP (to provide 5 µM final in the assay) in a Downloaded from

96-well plate. Test compound or vehicle was added to the wells, followed by

[35S]GTPγS to a final concentration of 0.2 nM. Plates were incubated for 45 min at room jpet.aspetjournals.org temperature and then centrifuged at 22ºC at 250g for 5 min prior to counting in a Wallac

MicroBeta® TriLux scintillation counter. Nonspecific binding was determined in the presence of 10 µM unlabelled GTPγS. The control agonist signal was determined in at ASPET Journals on September 24, 2021 wells containing vehicle in place of the test compound and the basal signal was determined in wells containing vehicle in place of both diluted compound and the RAMH challenge.

Data analysis for in vitro studies

Specific binding for the radioligand binding experiments was calculated by subtracting the mean of nonspecific binding obtained in each experiment from total binding. The percentage of specific binding was defined as individual specific binding/specific binding without test compound *100. Binding displacement and concentration response data were analyzed to determine IC50 or EC50 values by nonlinear regression using the dose- response equation (variable slope) in Prism 4.03 (GraphPad Software, Inc., San Diego,

CA): JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 12

Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*Hill Slope))

Ki or Kb,app values were calculated from the EC50 values.

Ex vivo autoradiography

CEP-26401 or vehicle (pH 2 water) was administered p.o. to Sprague-Dawley rats (male;

135-200 g; Charles River Laboratories, Wilmington, MA) 1 h prior to sacrifice by decapitation. Brains were removed and rapidly frozen in dry ice-cooled 2-methylbutane

(Sigma-Aldrich). Coronal sections were prepared on microscope slides and tested for Downloaded from

[3H]NAMH binding as previously described (Le et al., 2009). Briefly, dried slides were incubated with 0.3 nM [3H]NAMH in 50 mM sodium phosphate buffer (pH 7.4) at 23ºC jpet.aspetjournals.org for 30 min. The slides were washed, dried and prepared for image acquisition

(approximately 20 h) in a Beta-imager 2000 chamber using Beta Acquisition software

(Biospace, Paris, France). The radioligand signal in the region of interest was measured at ASPET Journals on September 24, 2021 in duplicate using the Beta vision software (Biospace) and average values were expressed as counts per minute per square millimeter (cpm/mm2). The inhibition of specific

[3H]NAMH binding ex vivo following administration of CEP-26401 was calculated relative to samples from vehicle-treated animals as follows: (1-(individual specific binding/average of vehicle binding))*100. Nonspecific binding was determined in the adjacent sections in the presence of 10 µM thioperamide. Specific binding was greater than 90% of total binding. Plasma and brain samples were collected from the study animals in the ex vivo binding experiments and were submitted for quantitative analysis to determine the respective compound concentrations as previously described (Hudkins et al., 2011).

Rat dipsogenia model JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 13

RAMH-induced water intake was measured in male Long Evans rats (> 300 g; Harlan,

Dublin, VA or Indianapolis, IN) for 30 min beginning 20 min after administration of

RAMH (10 mg/kg, i.p.). CEP-26401 (in saline) was administered p.o. at the indicated times prior to the initiation of the drinking trial period. Percent inhibition of RAMH- induced drinking was calculated for each rat based on normalization to the group mean of

RAMH-induced drinking using the following equation: [100 – (Dr /Dg RAMH)*100];

where Dr = the amount of water an individual rat drinks; and Dg RAMH = the group Downloaded from mean for the amount of water consumed by the RAMH-treated group. Group means for percent inhibition were then calculated for each dosage group together with the jpet.aspetjournals.org associated standard error of the mean. Treatment effects for percent inhibition of CEP-

26401 vs RAMH-induced dipsogenia were evaluated using a 1-way ANOVA (Prism 4).

Dunnett’s post hoc analysis was performed for multiple comparisons with the RAMH at ASPET Journals on September 24, 2021 group set as the control comparator.

Rat social recognition model of short term memory

The effect of CEP-26401 on short-term memory was determined in a rat social recognition model. Adult male rats (Sprague Dawley, 350-450 g; Charles River

Laboratories) were briefly exposed to a male juvenile rat (Sprague Dawley 80-130 g;

Charles River Laboratories) (Trial 1) and, after a varying inter-exposure intervals (IEI), the same juvenile rat was returned to the test box with the adult rat for a second exposure

(Trial 2). Following short IEIs (15-30 min), adult rats investigated the juvenile for significantly less time during Trial 2 relative to Trial 1, indicating that the adult rat retained memory for the juvenile. Memory of the juvenile was lost as the IEI was increased, with a 2 h IEI producing relatively equal exploration times on both the first JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 14 and second trials, resulting in a ratio of investigation duration (RID) near unity. A 2 h

IEI was therefore used to test putative memory-enhancing compounds. Rats were dosed with CEP-26401 or vehicle (pH 2 water) 30 min prior to Trial 2 for i.p. dosing and 120 min prior for p.o. dosing. Controls included separate groups of rats that received effective doses of compound and were subsequently exposed to a novel juvenile in Trial

2. Treatment effects on the RID were evaluated using 1-way ANOVAs (Prism 4).

Dunnett’s post hoc analysis was performed for multiple comparisons with the vehicle Downloaded from group.

Prepulse inhibition in mice jpet.aspetjournals.org Male DBA/2NCrl mice (19-27 g, 7-9 wks; Charles River Laboratories) were administered CEP-26401 or vehicle (saline) and risperidone or vehicle (5% dimethylsulfoxide (DMSO, Sigma-Aldrich) / 10% polyethylene glycol 400 (PEG400, at ASPET Journals on September 24, 2021

Hampton Research, Aliso Viejo, CA) / 85% sterile water) i.p. 30 min prior to testing.

The PPI procedure for light phase testing in SR-LAB™ ABS Startle Reflex Systems (San

Diego Instruments, San Diego, CA) was described previously (Flood et al., 2011).

Briefly, following a 5-min acclimation period of 65-dB background white noise, mice received 5 conditioning trials consisting of a startle pulse (120 dB, 40 ms). Mice then received trials in pseudorandom order (10 of each type) of the startle pulse alone, the startle pulse preceded by a prepulse (70, 75, 80, or 85 dB, 10 ms duration, and inter-pulse interval of 100 ms), or no stimulus. Activity was recorded for 65 ms. Inter-trial intervals ranged from 5 to 25 sec, averaging 15 sec. Maximum amplitudes (arbitrary mV readings) for each of the trial types were averaged as auditory response amplitudes. Percent PPI at each sound intensity was defined as [1-(prepulse plus startle response amplitude/ startle- JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 15 alone response amplitude)] x 100. Average percent PPI for each mouse was the mean percent PPI of all 4 prepulse sound intensities. Response amplitudes, after log transformation, and average percent PPI data were analyzed using ANOVAs. Post hoc pairwise comparisons versus the vehicle-treated group were made using Bonferroni t- tests. Compound exposures in plasma and brain for CEP-26401 and risperidone alone and in combination were assessed in DBA/2 mice at at the end of the PPI experiments

corresponding to 1 h after compound administrations, according to methods described Downloaded from previously (Flood et al., 2011).

Rat EEG sleep/wake studies jpet.aspetjournals.org Male Sprague Dawley rats (~350 g; Charles River Laboratories) surgically implanted for chronic recording of EEG (electroencephalographic) and EMG (electromyographic) signals were used to measure wake promoting activity as previously described (Le et al., at ASPET Journals on September 24, 2021

2008). An intraperitoneally-implanted transmitter (TA10TA-F40, Data Sciences

International, N. St. Paul, MN) was used to monitor motor activity. On the day of compound dosing, data were recorded for 24 h beginning 3 h after lights on. CEP-26401 in saline was administered 2 h after the start of recording. Sleep and wake activity was scored according to standard criteria as awake, rapid eye-movement (REM), or slow- wave or non-REM sleep according to visual analysis of EEG frequency and amplitude characteristics and EMG activity (Le et al., 2008). The time spent awake for individual

30 min time points after dosing was compared to corresponding vehicle group values using a Student’s t-test with significance set at p < 0.05. Also, the cumulative wake time at 4 h after dosing was evaluated using ANOVA followed by Dunnett’s post hoc test for treatment effects. Cumulative 4 h values of motor activity (arbitrary units) were JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 16 calculated, and motor intensity (motor activity normalized to EEG wake time) was calculated over the first 2 h post dosing as (average motor activity) / (average h awake by

EEG activity). Rebound hypersomnolence was evaluated by comparing total wake activity between drug and vehicle groups out to 22 h post dosing.

All experimental animal procedures were conducted in accordance with the National

Institute of Health guidelines and were approved by Cephalon’s Institutional Animal

Care and Use Committee. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 17

Results

In vitro characterization of CEP-26401 binding affinities

3 Binding of the radiolabeled H3R agonist, [ H]NAMH (West et al., 1990), was saturable in rat cortical membranes with a Kd value of 2.0 ± 0.03 nM (mean ± SEM) with the data being best fit to a single-site binding isotherm. The H3R antagonists, ciproxifan (Ligneau et al., 1998) and ABT-239 (Esbenshade et al., 2005), as well as the agonist, RAMH

3 (Arrang et al., 1987), displaced [ H]NAMH binding in rat cortical membranes (Table 1) Downloaded from as previously reported. CEP-26401 bound with high affinity (Ki = 2.7 ± 0.3 nM) to a single site (Hill slope = 1 ± 0.2). In membranes from cells recombinantly expressing the jpet.aspetjournals.org full length rH3R, binding affinities were similar to those determined against native rH3R in cortical membranes (Table 1). Unlike ciproxifan (Table 1), CEP-26401 demonstrated balanced affinities at recombinant rat and human H3R with apparent Ki values of 7.2 ± at ASPET Journals on September 24, 2021

0.4 nM and 2.0 ± 1.0 nM, respectively (Table 1) (Hudkins et al., 2011). Binding curves for the H3R antagonists in the recombinant systems were best fit by a one-site model, but average Hill slopes ranged from 0.70-0.89, suggesting the possible presence of a small fraction of low affinity sites for these ligands.

CEP-26401 demonstrated greater than 1000-fold selectivity for H3 versus recombinant human H1, H2 and H4 histamine receptors (Table 2) and against 165 additional GPCR, ion channel and enzyme targets (MDS Pharma Services, Taipei, Taiwan; data not shown).

Antagonist and inverse agonist activity in vitro

Antagonist potency was measured via inhibition of agonist-stimulated [35S]GTPγS binding in rat cortical membranes and in membranes expressing recombinant human or rat H3R (Table 3). The H3R agonist, RAMH, produced concentration dependent JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 18

35 increases in [ S]GTPγS binding with an EC50 = 52 ± 14 nM (n = 3) and a maximum stimulation of approximately 1.5-fold over basal in rat cortical membranes. The RAMH- induced response was inhibited by CEP-26401 with Kb,app = 4.5 ± 1.0 nM (Table 3), demonstrating potent antagonist activity at rH3R. Similarly, CEP-26401 potently

35 inhibited RAMH-induced [ S]GTPγS binding at recombinant rH3R (Kb, app = 1.0 ± 0.2 nM) and hH3R (Kb, app = 0.4 ± 0.1 nM) (Hudkins et al., 2011). Antagonism was reversed

by increasing concentrations of RAMH (data not shown). Ciproxifan and ABT-239 Downloaded from decreased basal [35S]GTPγS binding in a concentration-dependent manner in the recombinant systems (Table 3), consistent with known inverse agonist activity of these jpet.aspetjournals.org compounds (Esbenshade et al., 2005). CEP-26401 also decreased basal activity with

EC50 values of 2.0 ± 0.8 nM and 1.1 ± 0.0 nM for rat and human H3R, respectively (Table

3). The maximum reduction in basal [35S]GTPγS binding was similar for CEP-26401 at ASPET Journals on September 24, 2021

(300 nM), ciproxifan (10 µM) and ABT-239 (100 nM) at hH3R (78 ± 5%, 51 ± 20% and

67 ± 3%, n=3, respectively) (Figure 2A) and at rH3R (300 nM; 65 ± 4%, 63 ± 2% and 63

± 3%, n=3, respectively) (Figure 2B).

Inhibition of ex vivo [3H]NAMH binding

An ex vivo autoradiography assay was used to estimate H3R occupancy in rat cortex 1 h after p.o. administration of CEP-26401, as previously described (Le at al., 2009).

Specific binding of [3H]NAMH to rat cortical slices was identified by quantitative autoradiography in regions of H3R expression including striatum, cortex, hippocampus and thalamus (Pillot et al., 2002a). Specific binding of [3H]NAMH was decreased in a dose-dependent manner in cortical slices prepared from CEP-26401-treated animals with an OCC50 of 0.1 ± 0.0 mg/kg p.o. (Figure 3). Plasma and brain concentrations of CEP- JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 19

26401 measured in these animals 1 h following p.o. dosing at 0.1 mg/kg were 6.7 ± 1.0 ng/ml (22 nM) and 19.8 ± 3.8 ng/g (64 nM), respectively. These values are consistent with the previously reported pharmacokinetic properties of CEP-26401 in the rat, including high oral bioavailability (F = 83%) and good brain exposure (brain to plasma ratio of 2.6) (Hudkins et al., 2011).

CEP-26401 activity in the rat dipsogenia model

The H3R-selective agonist, RAMH, induces increased water intake that can be blocked by Downloaded from administration of H3R antagonists (Clapham and Kilpatrick, 1993; Fox et al., 2002). The dipsogenic effect is produced following i.p. or i.c.v. administration of RAMH, indicating jpet.aspetjournals.org interaction with central H3Rs (Lecklin et al., 1998). This model was used to demonstrate the pharmacological effects of CEP-26401 in vivo. CEP-26401 dose-dependently inhibited RAMH-induced dipsogenia when administered 15 min prior to RAMH with an at ASPET Journals on September 24, 2021

ED50 value of 0.06 (0.01-0.3) mg/kg, p.o. (Hudkins et al., 2011) demonstrating potent inhibition of the rH3R in vivo. Following a single dose of 1 mg/kg, p.o. CEP-26401,

RAMH-induced dipsogenia was significantly inhibited for 8 h with no measurable inhibition at 24 h post dose (Figure 4).

CEP-26401 activity in the rat social recognition model of short-term memory

In the social recognition model, CEP-26401 was effective at reducing the RID at doses over the range from 0.001-0.1 mg/kg, i.p. (Figure 5A) and at 0.01 and 0.1 mg/kg, p.o.

(Figure 5B), demonstrating potent enhancement of short-term sensory memory in this model. A separate group of rats treated with 0.1 mg/kg, i.p. of CEP-26401 were handled as described above, with a novel juvenile rat being presented for Trial 2 (0.1 + Novel;

Figure 5A). This group served as a control to determine whether CEP-26401 treatment JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 20 resulted in changes in investigative behavior independent of the juvenile memory paradigm. The investigation duration for this group showed not difference from vehicle- treated animals, indicating no change in the general investigative behavior of the animals.

CEP-26401 activity in the PPI model of sensorimotor gating

DBA/2 mice demonstrate naturally poor PPI that is reliably increased with antipsychotics and the H3R antagonists, ABT-239, thioperamide, and ciproxifan (Browman et al., 2004; Downloaded from

Fox et al., 2005; Flood et al., 2011). CEP-26401 administration in this model increased the average percent PPI following doses of 10 and 30 mg/kg, i.p. (Figure 6A). CEP- jpet.aspetjournals.org 26401 did not alter the amplitude of the startle reflex (Figure 6B). The , risperidone, also increased PPI at doses as low as 0.3 mg/kg, i.p. (Figure

6C). As observed elsewhere (Browman et al., 2004), the amplitude of the startle reflex at ASPET Journals on September 24, 2021 was reduced following administration of risperidone at 1 mg/kg, i.p. (data not shown).

CEP-26401 was then coadministered with a dose of risperidone that did not produce effects in this model as a single agent (0.1 mg/kg, i.p.). As in the single agent administration study, 10 mg/kg, i.p. CEP-26401 alone increased the average percent PPI and was not effective at a dose of 3 mg/kg, i.p. In contrast, the combination of 3 mg/kg

CEP-26401 and 0.1 mg/kg risperidone increased the average percent PPI (Figure 6D).

The combinations of 1–10 mg/kg, i.p. CEP-26401 with 0.1 mg/kg, i.p. risperidone did not alter no-stimulus and startle-alone amplitudes compared with both vehicles (data not shown). CEP-26401 and risperidone plasma and brain concentrations were determined in the DBA/2 mice and were not different when the compounds were dosed independently or coadministered (Table 4), suggesting the observed effects were not due to increased exposure to either compound in the coadministration paradigm. JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 21

CEP-26401 activity in rat EEG sleep/wake studies

CEP-26401 or vehicle was administered during the normal quiet period of the rat and wake, slow-wave sleep (SWS), and rapid-eye movement sleep (REMS) activities were compared across treatment groups. CEP-26401 increased wake activity and decreased

SWS and REMS in a dose-related manner for the first 4 h after dosing in the range tested

(3-30 mg/kg, p.o.; Figure 7A). At 30 mg/kg, CEP-26401 demonstrated robust wake promotion with the treated animals awake 90% of the time up to 3 h post dosing (Figure Downloaded from

7B). Following the period of significant wake enhancement (up to 3 h, 3.5 h and 5.5 h post dosing for 3, 10 and 30 mg/kg CEP-26401, respectively), the wake time for CEP- jpet.aspetjournals.org

26401 treated animals did not differ from that of vehicle-treated animals for several hours

(Figure 7B), indicating the absence of immediate sleep rebound. Furthermore, no

evidence of rebound hypersomnolence was observed up to 22 h after CEP-26401 at ASPET Journals on September 24, 2021 administration (Table 5). SWS and REMS onset latencies were also increased following administration of CEP-26401 (Table 6). Motor activity was unchanged, and hyperactivity (motor activity normalized to time spent awake) was absent during the periods of enhanced wake produced by CEP-26401. JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 22

Discussion

Several imidazole-containing H3R antagonists with high affinity for H3R in rat tissues, including ciproxifan, have low affinity at the human H3R (Lovenberg et al., 1999). CEP-

26401 had balanced affinity for both rat and human H3R and displayed greater than1000- fold selectivity for H3R over the other histamine receptors (Tables 1 and 2). H3R-induced effects are mediated via activation of Gi/o type G-proteins, making the binding of

[35S]GTPγS a sensitive measure of receptor activation (Clark and Hill, 1996; Rouleau et Downloaded from al., 2002). Consistent with high affinity binding, CEP-26401 demonstrated potent

35 antagonism of H3R agonist-induced [ S]GTPγS binding in the recombinant systems and jpet.aspetjournals.org in rat brain membranes. Although inverse agonist activity in rat brain preparations has been reported for some functional measures of H3R activity (Morisset et al., 2000), only a

35 small fraction of basal [ S]GTPγS binding is modified by H3R activity in rat brain at ASPET Journals on September 24, 2021 membranes (Clark and Hill, 1996; Rouleau et al., 2002). Membranes from recombinant

H3R-expressing cells were used to determine inverse agonist activity for CEP-26401 and reference compounds with CEP-26401, ciproxifan and ABT-239 demonstrating potent inverse agonist activity. The relevance of inverse agonist activity demonstrated in vitro, to the function of H3R in physiological or pathological conditions in vivo is not clear due to the lack of neutral antagonists.

In vivo H3R occupancy was estimated using autoradiographic ex vivo radioligand binding in rat cortical slices following oral administration of CEP-26401 that avoided the potential to underestimate receptor occupancy reported with ex vivo binding studies in tissue homogenates (Le et al., 2009). ID50 values for reference compounds were similar to those determined using an in vivo radiotracer to measure H3R occupancy (Le et al., JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 23

2009; Miller et al., 2009). The OCC50 of 0.1 ± 0.0 mg/kg, p.o. observed for CEP-26401

(Figure 3) was attained at a total brain concentration of 64 nM, 20-times higher than the binding Ki value of 2.7 nM determined in rat brain homogenates, in vitro. While CEP-

26401 displayed low protein binding in dialysis studies with rat plasma proteins and

~40% free fraction when dialysed with rat brain homogenate (Hudkins et al., 2011), these in vitro measures are estimates and may not reflect the actual free fraction in vivo. In

addition, the preparation of brain homogenates and the presence of assay buffer Downloaded from components may affect binding affinities determined in vitro. CEP-26401 potently inhibited RAMH-induced drinking (ED50 of 0.06 mg/kg, p.o.) in the rat dipsogenia jpet.aspetjournals.org model, in agreement with the OCC50 determined by ex vivo binding. This agreement may reflect the similar nature of inhibiting the pharmacological effect of RAMH in the dipsogenia model, compared to displacement of an agonist radioligand in the ex vivo at ASPET Journals on September 24, 2021 binding assay. The efficacy and duration of activity in the dipsogenia model (Figure 4) were consistent with the rat plasma half-life of CEP-26401 (2.5 ± 0.3 h) (Hudkins et al.,

2011).

The expression of H3R in brain regions involved in memory, attention and executive function (Pillot et al., 2002a; Martinez-Mir et al., 1990) along with the role of H3R in modulating neurotransmitter release (Esbenshade 2008) led to the evaluation of H3R antagonists for potential clinical utility in the treatment of cognitive disorders associated with a number of disease states (Brioni et al., 2011). The rat social recognition model measures short-term memory (Thor and Holloway, 1982) and a number of memory- enhancing compounds with diverse mechanisms are active in this model including H3R antagonists (Prast et al., 1996; Fox et al., 2005). CEP-26401 demonstrated potent activity JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 24

in this model without disrupting normal exploratory behaviors. While the estimated H3R occupancy at effective doses of CEP-26401 was low, rodent models of short-term memory are particularly sensitive to the effects of H3R antagonists (Fox et al., 2005;

Medhurst et al., 2007) with a small percentage occupancy (as low as < 10%) producing efficacy in cognition models (Miller et al., 2009).

Effects in preclinical models of PPI suggest that compounds of various classes, including

H3R antagonists may have utility in the treatment of schizophrenia (Browman et al., Downloaded from

2004; Fox et al., 2005; Flood et al., 2011). However, while the DBA/2 mouse PPI model is sensitive to H3R antagonists, other preclinical models relevant to schizophrenia do not jpet.aspetjournals.org robustly respond to H3R antagonists (Burban et al., 2010; Flood et al., 2011). Although

CEP-26401 effectively increased the PPI response in DBA/2 mice, fully efficacious doses

(Figure 6) were higher than those effective in the rat social recognition model (Figure 5). at ASPET Journals on September 24, 2021

When coadministered with the antipsychotic, risperidone, a lower dose of CEP-26401 (3 mg/kg , i.p.) was active in this model, lowering the effective dose of risperidone from 0.3 to 0.1 mg/kg, i.p. Previous studies suggested increased efficacy of in a rodent catalepsy model following coadministration of the imidazole-based H3R antagonist, ciproxifan (Pillot et al., 2002b). However ciproxifan inhibited cytochrome P450 (CYP) enzymes with the coadministration decreasing haloperidol metabolism, complicating interpretation of these studies (Zhang et al., 2005). Unlike ciproxifan, CEP-26401 does not inhibit CYP enzymes (Hudkins et al., 2011) and the plasma and brain concentrations of CEP-26401 and risperidone were unaltered by coadministration. As an adjunctive therapy, CEP-26401 could thus result in effective treatment of schizophrenia at lower doses of the antipsychotic with correspondingly lower incidence of side effects. JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 25

Treatment with CEP-26401 may also address the cognitive deficits associated with schizophrenia that are poorly controlled by currently used drugs. Antipsychotics interact with a broad spectrum of receptors, including the H1R. H3R antagonism would be expected to increase histamine release resulting in increased H1R activation. The effects of such interactions on the efficacy and side effect profile of antipsychotics coadministered with H3R antagonists should be considered in future studies.

H3R antagonists increase wake in several species, albeit at dose ranges higher than those Downloaded from demonstrating efficacy in models of enhanced cognition. The maintained wake level was limited to 50-60% time awake, compared to baseline activity of 25-30% wake (Le et al., jpet.aspetjournals.org 2008). Robust wake promotion (85% and 188% increases over vehicle in the 4 h following oral dosing) was produced by CEP-26401 at 10 and 30 mg/kg p.o., respectively

(Figure 7A). The enhanced wake was not accompanied by increased motor activity or at ASPET Journals on September 24, 2021 intensity, or sleep rebound, an effect similar to that seen with modafinil but distinct from that of stimulants like amphetamine (Parmentier et al., 2007).

Wake promoting doses of CEP-26401 and those doses active in the PPI model produced full occupancy of cortical H3R as determined via the ex vivo occupancy study (Figure 3), while the doses active in the social recognition model correspond to lower receptor occupancy. This difference has been demonstrated for a number of H3R antagonists and may reflect effects on different brain regions and neurotransmitters. For example, the

H3R antagonist, ABT-239, increased cortical ACh release at doses as low as 0.1 mg/kg i.p., while cortical dopamine release was increased only at 3 mg/kg (Fox et al., 2005).

Distinct effects on neurotransmitters in different brain regions were described with the

H3R antagonist, GSK189254 which increased ACh release in the anterior cingulate cortex JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 26 at 1 mg/kg and at 0.3 mg/kg in the dorsal hippocampus (Medhurst et al., 2007). These regional and neurotransmitter-selective effects may be reflected in the differential potencies seen in models of short term memory, which may be dependent on ACh, versus models in which modulation of dopamine levels is thought to play a key role, such as

PPI. In pilot clinical trials the H3R antagonist, tiprolisant (BF2.649), decreased daytime sleepiness in narcoleptics (22 patients) (Lin et al., 2008) confirming a potential role for

H3R antagonists in treating narcolepsy. MK-0249 increased sleep latency in 24 healthy Downloaded from subjects following a period of sleep deprivation (Iannone et al., 2010). The effects on sleep and vigilance in early clinical trials suggest a role for H3R antagonists in treating jpet.aspetjournals.org excessive daytime sleepiness resulting from a variety of causes and indicate that rodent models may underestimate the wake promoting effects of this class of compounds in man. CEP-26401 was well tolerated up to 100 mg/kg, p.o. in the rat Irwin behavioral at ASPET Journals on September 24, 2021 battery (Hudkins et al., 2011) providing a large estimated therapeutic index (TI) in comparison to the active dose of 0.01 mg/kg, p.o. in the social recognition model and a TI greater than 33 in comparison to the wake promoting dose of 3 mg/kg, p.o. in the rat.

The present data demonstrate potent pharmacological effects following oral dosing of

CEP-26401, a compound with favorable drug-like properties (Hudkins et al., 2011) and suggest that this novel H3R antagonist may have therapeutic utility in the treatment of cognitive and attentional disorders. In addition to tiprolisant and MK-0249, several H3R antagonists including ABT-288, AZD-5213, JNJ-31001074, PF-03654746, GSK239512 and MK-3134 have advanced to clinical trials for the treatment of sleep disturbances and/or cognitive disorders associated with AD, ADHD, narcolepsy or schizophrenia

(Brioni et al., 2011; Leurs et al., 2011). Results from the clinical trials with H3R JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 27 antagonists are eagerly awaited to establish the potential for novel CNS active drugs with advantages over the currently used stimulants, nootropics and/or antipsychotics.

Acknowledgements

The authors would like to thank Robert Bendesky, Amy DiCamillo, James Finn, Beverly

Holskin, Yin-Guo Lin, Jacquelyn Lyons and Val Marcy for their excellent technical

contributions, Mehran Yazdanian and Sheryl Meyer for helpful discussion of the work Downloaded from and comments on the manuscript and Jeff Vaught, Joe Turi and the late Frank Baldino for their support. MW would like to acknowledge the many contributions of Clark Tedford, jpet.aspetjournals.org the late Art Hancock, Tim Esbenshade, Yusef Bennani and Tim Lovenberg to advancing the field of H3R antagonist research.

at ASPET Journals on September 24, 2021

Authorship Contributions

Participated in research design: Raddatz, Hudkins, Mathiasen, Gruner, Flood, Aimone,

Le, Schaffhauser, Gasior, Bozyczko-Coyne, Marino, Ator, Bacon, Mallamo and Williams

Conducted experiments: Raddatz, Hudkins, Mathiasen, Gruner, Flood, Aimone and Le

Contributed new reagents or analytic tools: Hudkins

Performed data analysis: Raddatz, Hudkins, Mathiasen, Gruner, Flood, Aimone, Le,

Schaffhauser, Bozyczko-Coyne and Marino

Wrote or contributed to the writing of the manuscript: Raddatz, Mathiasen, Gruner,

Flood, Aimone, Le, Schaffhauser, Gasior, Bozyczko-Coyne, Marino, Ator and Williams JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 28

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Footnotes

*Current addresses: JRM: Taconic, Cranbury, NJ JAG: Melior Discovery, Exton, PA DGF: EnVivo Pharmaceuticals, Inc., Watertown, MA SL : WuXi AppTec (Shanghai) Co., Ltd., Shanghai, CN HS: CNS Research, Roche, Basel, CH MG: Discovery Medicine Neuroscience, Bristol-Myers Squibb, Princeton, NJ MJM: CNS Research, Merck Research Laboratories, West Point, PA MW: Northwestern University, Chicago, IL Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

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Figure Legends

Figure 1. Structures. Structure of CEP-26401 (Hudkins et al., 2011) and ABT-239

(Cowart et al., 2004).

Figure 2. Inverse agonist activity at recombinant human and rat H3R. CEP-26401

(filled squares), ciproxifan (open triangles) and ABT-239 (open diamonds) concentration- Downloaded from dependently inhibited basal [35S]GTPγS binding in recombinant human (A) and rat (B)

H3R membranes. Membranes were incubated for 45 min with SPA beads, 0.2 nM jpet.aspetjournals.org [35S]GTPγS and increasing concentrations of test compounds or vehicle (to determine basal activity). Nonspecific binding was determined in the presence of 10 µM GTPγS.

(Mean ± SD, n = 3). at ASPET Journals on September 24, 2021

Figure 3. Dose-dependent inhibition of [3H]NAMH ex vivo binding in rat cortical slices. Rats were treated with vehicle (pH 2 water) or CEP-26401 at the indicated doses, sacrificed after 1 h and cortical slices were prepared for radioligand binding. CEP-26401

3 dose-dependently inhibited [ H]NAMH ex vivo binding with an OCC50 value of 0.1 ± 0.0 mg/kg, p.o. (n = 6 for each dose in three independent experiments).

Figure 4. Duration of activity of CEP-26401 in the rat dipsogenia model. Rats were treated with vehicle (saline) or CEP-26401 at 1 mg/kg, p.o. at the indicated times prior to initiation of water drinking. RAMH was administered 20 min before the water drinking JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 37 period of 30 min. * p<0.05 (ANOVA, Dunnett’s post hoc) from vehicle / RAMH treated.

(Mean ± SEM, n = 10-20/ group).

Figure 5. CEP-26401 activity in the social recognition model of short-term memory.

CEP-26401 activity was evaluated in the social recognition model following i.p. (A) or p.o. (B) dosing. Vehicle (Veh, pH 2 water) or CEP-26401 was administered 30 min prior to Trial 2 for i.p. and 120 min for oral dosing. In A, the group indicated by 0.1 + Novel Downloaded from was administered 0.1 mg/kg, i.p. CEP-26401 and was exposed to a novel juvenile rat in

Trial 2 as a control for compound effects on investigative behavior. Significant effects at jpet.aspetjournals.org 0.001 and 0.01 mg/kg, i.p. were demonstrated in a replicate study (data not shown). *p <

0.05 (ANOVA, Dunnett’s post hoc test) vs. vehicle. (Mean ± SEM; i.p., n = 12-20 /

group; p.o., n = 14 / group). at ASPET Journals on September 24, 2021

Figure 6. Increases in PPI in the DBA/2 mouse model. Vehicle (V), CEP-26401 (A-B) or risperidone (C) was administered i.p. at the indicated doses. The percent PPI averaged across prepulse intensities and startle amplitudes were determined. In 2-way repeated measures ANOVAs, the compound treatment and prepulse intensity effects were significant for percent PPI but the interactions were not significant. Log10 transformed startle amplitudes were analyzed by 1-way ANOVAs. Asterisks represent significant post hoc compound treatment differences from vehicle: *p < 0.05, **p < 0.01 and ***p <

0.001. (Mean + SEM, n = 20 / group). (D) CEP-26401 was tested at the indicated doses with coadministration of either risperidone (0.1 mg/kg, i.p.) or vehicle (5% DMSO / 10%

PEG 400). The overall 2-way ANOVA was significant for the effect of CEP-26401 (p < JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 38

0.001) and for the effect of risperidone (p = 0.005). Post hoc comparisons with the appropriate control groups (indicated by arrowheads and the comparator symbol) were made using Bonferroni t-tests. CEP-26401 alone at 10 mg/kg, i.p. differed from the group administered both vehicles (**p < 0.01). Both 3 and 10 mg/kg, i.p. CEP-26401 in combination with 0.1 mg/kg, i.p. risperidone differed compared with the group administered 0.1 mg/kg, i.p. risperidone alone (^^^p < 0.001). CEP-26401 at 3 mg/kg,

i.p. coadministered 0.1 mg/kg, i.p. risperidone differed from 3 mg/kg, i.p. CEP-26401 Downloaded from alone (+p<0.05). (Mean + SEM, n = 29-30 / group).

jpet.aspetjournals.org Figure 7. CEP-26401-induced wake promotion. CEP-26401 (saline as vehicle) was administered p.o. to male rats chronically implanted with electrodes for recording EEG and EMG activity. (A) Cumulative wake, slow wave sleep (SWS), and rapid eye at ASPET Journals on September 24, 2021 movement sleep (REMS) 4 h AUC values shown for each dose (Mean + SEM, n = 7-8 / group). * p < 0.05, Dunnett’s test vs. vehicle (Veh). (B) Dose-related increases in time awake (mean + SEM) were observed at 3, 10, and 30 mg/kg, p.o. (n = 8, 8, and 6, respectively). Significant difference from vehicle (n = 10) for each 0.5 h time point is shown along top of graph for each dose; * p < 0.05, unpaired t-test.

JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 39

Tables

Table 1. [3H]NAMH binding displacement affinities for CEP-26401 and reference ligands at rat and human H3R

Rat Cortical Recombinant rH3R Recombinant hH3R

Membrane Ki (nM) Ki (nM) Ki (nM)

CEP-26401 2.7 ± 0.3 7.2 ± 0.4 2.0 ± 1.0 Downloaded from

Ciproxifan 1.4 ± 0.3 1.3 ± 0.6 85.9 ± 9.8

ABT-239 15.6 ± 3.1 31.2 ± 5.4 9.1 ± 2.8 jpet.aspetjournals.org RAMH 1.7 ± 0.4 1.7 ± 0.5 1.9 ± 0.4

Mean ± SD, n = 3-6

at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 40

Table 2. Inhibition of binding at histamine receptor subtypes by CEP-26401 (10 µM)

Assay % inhibitiona

b hH1R Binding 10.6 ± 12.7

c hH2R Binding 4.1 ± 9.9

d hH4R Binding -1.3 ± 7.8

a Mean ± SD, n = 3

b [3H]pyrilamine binding Downloaded from

c [3H]tiotidine binding

d [3H]histamine binding jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 41

Table 3. Functional potencies of CEP-26401 and reference compounds in the

[35S]GTPγS binding assay

Antagonist Activity Inverse Agonist Activity

Kb, app (nM) EC50 (nM)

Rat Cortical Recombinant Recombinant Recombinant Recombinant

Membrane rH3R hH3R rH3R hH3R Downloaded from

CEP-26401 4.5 ± 1.0 1.0 ± 0.2 0.4 ± 0.1 2.0 ± 0.8 1.1 ± 0.0

Ciproxifan 0.1 ± 0.05 0.8 ± 0.3 54 ± 31 1.2 ± 0.3 188 ± 83 jpet.aspetjournals.org ABT-239 1.0 ± 0.8 4.5 ± 2.3 0.6 ± 0.2 5.8 ± 2.2 2.7 ± 1.0

Mean ± SD, n = 3-5

at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 42

Table 4. Plasma and brain concentrations of CEP-26401 and risperidone in DBA/2 mice 1 h following coadministration

Doses CEP-26401 concentrationa Risperidone concentrationa

CEP-26401 Risperidone Plasma Brain Plasma Brain

(mg/kg, i.p.) (mg/kg, i.p.) (ng/ml) (ng/g) (ng/ml) (ng/g)

1 Vehicle 59 ± 3 137 ± 7

3 Vehicle 192 ± 8 447 ± 24 Downloaded from

10 Vehicle 791 ± 51 1761 ± 76

Vehicle 0.1 4.4 ± 0.2 6.9 ± 0.9 jpet.aspetjournals.org 1 0.1 60 ± 4 136 ± 8

3 0.1 180 ± 7 433 ± 23 4.0 ± 0.1 4.9 ± 0.4

10 0.1 691 ± 21 1720 ± 78 at ASPET Journals on September 24, 2021

aMean ± SEM, n = 5-12 per group

JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 43

Table 5. Lack of sleep rebound following CEP-26401-induced wake promotion

Wake Times (min/h)a

CEP-26401 dose Matching time (mg/kg p.o.) CEP-26401 treated period for vehicle treated 3 34.8 ± 1.4 33.7 ± 1.2

10 32.5 ± 0.7 34.1 ± 1.2 Downloaded from

30 34.8 ± 1.6 35.1 ± 1.2 aMean ± SEM, n = 6-10; wake times measured from end of period of enhanced wake (3 jpet.aspetjournals.org h, 3.5 h and 5.5 h post dosing for 3, 10 and 30 mg/kg CEP-26401, respectively) to 22 h post dosing. p > 0.05 by Student’s t-test comparing each dose to the appropriate time period for the vehicle-treated group. at ASPET Journals on September 24, 2021

JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version.

JPET #186585 44

Table 6. Effect of CEP-26401 on sleep latencies and motor activity following oral dosing

Parameter Vehicle 3 mg/kg 10 mg/kg 30 mg/kg

SWS latencya (min) 32.9 ± 4.2 38.1 ± 5.4 82.7 ± 17.7* 216.7 ± 23.0*

REMS latencya (min) 42.5 ± 3.5 86.0 ± 27.9 113.9 ± 13.2 281.6 ± 44.9*

Motor activityb 4.74 ± 1.30 5.47 ± 0.72 6.93 ± 1.16 7.92 ± 1.38

Motor intensityc 2.94 ± 0.75 2.73 ± 0.52 2.28 ± 0.35 2.37 ± 0.36 Downloaded from

Mean ± SEM. n = 6-10 per group a p < 0.05, ANOVA for treatment, * p < 0.05 vs. vehicle by Dunnett’s test. jpet.aspetjournals.org bCumulative motor activity for 4 h post dosing, arbitrary units. cMotor intensity = (average motor activity units for 2 h post dosing) / (average EEG wake

time (h) for 2 h post dosing). at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021 JPET Fast Forward. Published on October 14, 2011 as DOI: 10.1124/jpet.111.186585 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021