Volume 156, Issue 8 p. 1326-1341
Free Access

Biochemical and behavioural characterization of EMPA, a novel high-affinity, selective antagonist for the OX2 receptor

P Malherbe

P Malherbe

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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E Borroni

E Borroni

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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L Gobbi

L Gobbi

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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H Knust

H Knust

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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M Nettekoven

M Nettekoven

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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E Pinard

E Pinard

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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O Roche

O Roche

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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M Rogers-Evans

M Rogers-Evans

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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JG Wettstein

JG Wettstein

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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J-L Moreau

J-L Moreau

Discovery Research CNS, F. Hoffmann-La Roche Ltd., Basel, Switzerland

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First published: 06 April 2009
Citations: 72
Dr P Malherbe, F. Hoffmann-La Roche Ltd., Psychiatry Disease Area, Bldg. 69/333B, CH-4070 Basel, Switzerland. E-mail: [email protected]


Background and purpose: The OX2 receptor is a G-protein-coupled receptor that is abundantly found in the tuberomammillary nucleus, an important site for the regulation of the sleep-wake state. Herein, we describe the in vitro and in vivo properties of a selective OX2 receptor antagonist, N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide (EMPA).

Experimental approach: The affinity of [3H]EMPA was assessed in membranes from HEK293-hOX2-cells using saturation and binding kinetics. The antagonist properties of EMPA were determined by Schild analysis using the orexin-A- or orexin-B-induced accumulation of [3H]inositol phosphates (IP). Quantitative autoradiography was used to determine the distribution and abundance of OX2 receptors in rat brain. The in vivo activity of EMPA was assessed by reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion during the resting phase in mice and the reduction of spontaneous locomotor activity (LMA) during the active phase in rats.

Key results: [3H]EMPA bound to human and rat OX2-HEK293 membranes with KD values of 1.1 and 1.4 nmol·L−1 respectively. EMPA competitively antagonized orexin-A- and orexin-B-evoked accumulation of [3H]IP at hOX2 receptors with pA2 values of 8.6 and 8.8 respectively. Autoradiography of rat brain confirmed the selectivity of [3H]EMPA for OX2 receptors. EMPA significantly reversed [Ala11,D-Leu15]orexin-B-induced hyperlocomotion dose-dependently during the resting phase in mice. EMPA, injected i.p. in rats during the active phase, reduced LMA dose-dependently. EMPA did not impair performance of rats in the rotarod procedure.

Conclusions and implications: EMPA is a high-affinity, reversible and selective OX2 receptor antagonist, active in vivo, which should prove useful for analysis of OX2 receptor function.


  • [Ca2+]i
  • intracellular calcium concentration
  • CHO
  • Chinese hamster ovary
  • CSF
  • cerebrospinal fluid
  • EMPA
  • N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide
  • Fluorometric Imaging Plate Reader
  • GPCRs
  • G-protein-coupled receptors
  • IP
  • inositol phosphates
  • NREM
  • non-REM
  • REM
  • rapid eye movement
  • RT
  • reverse transcriptase
  • Introduction

    The orexins/hypocretins, a family of hypothalamic neuropeptides, play an important role in modulating feeding behaviour, energy homeostasis and in regulating the sleep-wake cycle (Siegel, 2004; Ohno and Sakurai, 2008). The two members of the family, orexin-A/hypocretin-1 (33 amino acids) and orexin-B/hypocretin-2 (28 amino acids), are derived from the same precursor by proteolytic processing of the 130-amino-acid polypeptide prepro-orexin (de Lecea et al., 1998; Sakurai et al., 1998). Two receptor subtypes, termed OX1 and OX2, have been identified (nomenclature follows Alexander et al., 2008). Characterization in binding and functional assays demonstrated that orexin-A is a non-selective neuropeptide that binds with similar affinities to OX1 and OX2 receptors, while orexin-B is selective and has a 10-fold higher affinity for OX2 over OX1 receptors (Sakurai et al., 1998). Both receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) that couple to Gq/11 and contribute to the activation of phospholipase C, leading to the elevation of intracellular Ca2+ concentrations, [Ca2+]i (Sakurai et al., 1998). In addition, a detailed signalling profile of human OX2 receptors has recently shown that these receptors couple to Gs as well as Gq/11 and Gi pathways (Tang et al., 2008). Northern blot analysis of adult rat tissues showed that prepro-orexin mRNA is detected exclusively in the brain, except for a small amount in the testis, and that OX1 and OX2 receptor transcripts are exclusively detected also in the brain (Sakurai et al., 1998). The expression of orexins and their receptors have been detected by RT-PCR and immunohistochemistry in peripheral tissue including intestine, pancreas, adrenals, kidney and reproductive tract (Voisin et al., 2003; Heinonen et al., 2008). Distribution studies in rat brain using in situ hybridization and immunohistochemistry have shown that orexin neurons are found only in the lateral hypothalamic area yet having projections into the entire CNS (Peyron et al., 1998; Nambu et al., 1999). Although OX1 and OX2 receptors are present in most brain regions, OX1 receptors are most abundantly expressed in the locus coeruleus while OX2 receptors are expressed in regions controlling arousal, especially in the tuberomammillary nucleus, an important site for the regulation of sleep and wakefulness (Marcus et al., 2001). The high expression of orexin receptors in brain regions such as neocortex L6, ventral tegmental area, locus coeruleus, preoptic area, dorsal and medial raphe nuclei and periaqueductal area, in combination with the projections of the hypothalamic orexin-containing neurons towards limbic and brain stem structures also are consistent with a crucial role for the orexin system in the complex regulation of emotional responses (Marcus et al., 2001).

    The disruption of orexin signalling is thought to be the cause of narcolepsy, an assumption based on several lines of evidence: prepro-orexin knockout (KO) mice presented a phenotype with characteristics remarkably similar to narcolepsy (Chemelli et al., 1999); a mutation (canarc-1) that disrupts the gene encoding for OX2 receptors was found to be responsible for canine narcolepsy (Lin et al., 1999); a lack of orexin-A and orexin-B was observed in human narcoleptic patients (Nishino et al., 2000; Peyron et al., 2000); and it has been shown that modafinil, an anti-narcoleptic drug with unknown mechanism of action, activates orexin neurons (Chemelli et al., 1999). Intracerebroventricular (i.c.v.) administration of orexin-A was shown to dose-dependently increase the wake time and reduce total rapid eye movement (REM) sleep by 84% in rats (Piper et al., 2000). Wakefulness induced by orexin-A is likely to be mediated by the histaminergic system through OX2 receptors and is almost completely absent in H1 receptor KO mice (Huang et al., 2001; Yamanaka et al., 2002). Interestingly, a non-selective H1 antagonist pyrilamine attenuated the effect of i.c.v. injected orexin-A on wakefulness in rats (Yamanaka et al., 2002). OX2 receptor KO mice exhibit abnormal attacks of non-REM (NREM) sleep and marked sleep-wake fragmentation along with mild cataplexy (Willie et al., 2003). Taken together, these observations are consistent with a vital role for the orexin system, and especially OX2 receptors, in the modulation of sleep. Indeed, recent preclinical (dog and rat) and phase I (healthy male subjects; single dose) investigations have shown that the dual OX1/OX2 receptors antagonist almorexant (ACT-078573) promoted sleep (NREM and REM) in animals and humans without disrupting sleep architecture, an action that validated the notion that OX receptor antagonists could be effective hypnotics for the treatment of insomnia (Brisbare-Roch et al., 2007). Moreover, the orexin neuronal system has also been implicated in pain modulation within the CNS (Holland and Goadsby, 2007). The genetic linkage and haplotype analyses have shown an association between the OX2 receptor gene and cluster headaches (Rainero et al., 2007; 2008). The G1246A polymorphism (substitution of valine 308 by isoleucine) of the OX2 gene has been suggested to modulate the genetic risk for cluster headaches by interfering with the dimerization process of OX2 receptors (Rainero et al., 2008).

    Given the diverse functioning of orexin systems, selective antagonists targeted at OX1 or OX2 receptors can provide crucial tools for deciphering and understanding the physiological and pathophysiological roles of each receptor subtype. SB-334867-A, (1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride) was the first non-peptide OX1 receptor antagonist reported (Smart et al., 2001). SB-334867-A has been intensively used for in vivo studies of orexin-A physiological effects (Nishino, 2007). N-(4-pyridylmethyl)(S)-tert-leucyl-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline was the first non-peptide selective OX2 receptor antagonist to be described (Hirose et al., 2003). Until now, there was a lack of selective radioligands for OX2 receptors. In the current study, the binding characteristics of the selective OX2 receptor antagonist radioligand, [3H]N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide ([3H]EMPA), to cell membranes from HEK293 cells expressing hOX2 receptors and to rat brain sections are described. Data show that EMPA is a potent, highly selective and reversible antagonist of OX2 receptors and that it displays in vivo activity in the reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion in mice and a decrease of spontaneous locomotion during the active phase in rats.


    Plasmids, cell culture and membrane preparation

    cDNA encoding human OX2 (Accession No. O43614), rat OX2 (Accession No. P56719) and human OX1 receptors (Accession No. O43613) were subcloned into pCI-Neo expression vectors (Promega, Madison, WI). HEK293 cells were transfected as previously described (Malherbe et al., 2006). After 48 h post-transfection, cells were harvested and washed 3 times with cold PBS and frozen at −80°C. The pellet was suspended in ice-cold buffer containing 15 mmol·L−1 Tris-HCl, pH 7.5, 2 mmol·L−1 MgCl2, 0.3 mmol·L−1 EDTA, 1 mmol·L−1 EGTA, protease inhibitor cocktail EDTA-free (Cat. No. 11 873 580 001, Roche Applied Science, RAS, Rotkreuz, Switzerland) and homogenized with a Polytron (Kinematica AG, Basel, Switzerland) for 30 s at 16 000 r.p.m. After centrifugation at 48 000×g for 30 min at 4°C, the pellet was suspended in ice-cold buffer containing 75 mmol·L−1 Tris-HCl, pH 7.5, 12.5 mmol·L−1 MgCl2, 0.3 mmol·L−1 EDTA, 1 mmol·L−1 EGTA, 250 mmol·L−1 sucrose, protease inhibitor cocktail EDTA-free. After homogenization for 15 s at 16 000 r.p.m., protein content was measured using the BCA method (Pierce, Socochim, Lausanne, Switzerland) with bovine serum albumin as the standard. The membrane homogenate was frozen at −80°C before use.

    [3H]EMPA binding

    After thawing, membrane homogenates were centrifuged at 48 000×g for 10 min at 4°C, pellets were re-suspended in the binding buffer (25 mmol·L−1 HEPES, pH 7.4, 1 mmol·L−1 CaCl2, 5 mmol·L−1 MgCl2, 0.5% BSA, 0.05% Tween 20) to a final assay concentration of 2.5 µg protein per well. Saturation isotherms were determined by the addition of various concentrations of [3H]EMPA to these membranes (in a total reaction volume of 500 µL) for 60 min at 23°C. At the end of incubation, membranes were filtered onto unitfilter, a 96-well white microplate with bonded GF/C filter pre-incubated 1 h in wash buffer (25 mmol·L−1 HEPES, pH 7.4, 1 mmol·L−1 CaCl2, 5 mmol·L−1 MgCl2) plus 0.5% polyethylenimine, with a Filtermate 196 harvester (PerkinElmer Life and Analytical Sciences, Waltham, MA) and washed 4 times with ice-cold wash buffer. Non-specific binding (NSB) was measured in the presence of 10 µmol·L−1 EMPA. Radioactivity on the filter was counted (5 min) on a Top-Count microplate scintillation counter (PerkinElmer Life and Analytical Sciences) with quenching correction after addition of 45 µL of microscint 40 (PerkinElmer Life and Analytical Sciences) and shaking for 1 h.

    Saturation experiments were analysed by Prism 4.0 (GraphPad software, San Diego, CA) using the rectangular hyperbolic equation derived from the equation of a bimolecular reaction and the law of mass action, B = (Bmax*[F])/(KD+[F]), where B is the amount of ligand bound at equilibrium, Bmax is the maximum number of binding sites, [F] is the concentration of free ligand and KD is the ligand dissociation constant. For inhibition experiments, membranes were incubated with [3H]EMPA at a concentration equal to the KD value of radioligand and 10 concentrations of the inhibitory compound (0.0001–10 µmol·L−1). IC50 values were derived from the inhibition curve and the affinity constant (Ki) values were calculated using the Cheng-Prussoff equation Ki= IC50/(1 +[L]/KD) where [L] is the concentration of radioligand and KD is its dissociation constant at the receptor, derived from the saturation isotherm. To measure association kinetics, membranes were incubated at 23°C in the presence of radioligand (∼1.1 nmol·L−1[3H]EMPA) for 0, 1, 3, 5, 7, 10, 15, 20, 30, 60, 90 or 120 min, then terminated by rapid filtration. Dissociation kinetics were measured by adding at different times before filtration, 10 µmol·L−1 EMPA to membranes pre-incubated at 23°C for 1 h in the presence of ∼1.1 nmol·L−1[3H]EMPA. Binding kinetics parameters, Kob and Koff values (observed on and off rates), were derived from association-dissociation curves using the one phase exponential association and decay equations (Prism 4.0, GraphPad software) respectively. Kon, half-life and Kd were calculated using the Kon= (Kob− Koff)/[ligand], t1/2= ln2/K and KD= Koff/Kon equations respectively.

    Accumulation of [3H]inositol phosphates (IP)

    Accumulation of [3H]IP was measured as described previously (Malherbe et al., 2006) with the following adaptations. The Chinese hamster ovary (CHO) (dHFr-) mutant cell line stably expressing human OX2 receptors, CHO(dHFr-)-hOX2, was maintained in Dulbecco's modified Eagle's medium (DMEM; 1×) with GlutaMaxTM1, 4500 mg·L−1 D-glucose and sodium pyruvate, 5% dialysed fetal calf serum, 100 µg·mL−1 penicillin and 100 µg·mL−1 streptomycin (Pen/Strep). Cells were washed twice in labelling medium: DMEM without inositol (MP Biomedicals, Irvine, CA), 10% dialysed FCS, 1% Pen/Strep, 2 mmol·L−1 glutamate. Cells were seeded at 8 × 104 cells per well in poly-D-lysine-treated 96-well plates in the labelling medium supplemented with 5 µCi·mL−1 of myo-[1,2-3H]-inositol and were incubated overnight. The following day, cells were washed 3 times with the wash buffer (1 × HBSS, 20 mmol·L−1 HEPES, pH 7.4) and then incubated for 10 min at 23°C in assay buffer (1 × HBSS, 20 mmol·L−1 HEPES, pH 7.4, 0.1% BSA, plus 8 mmol·L−1 LiCl to prevent phosphatidyl-inositide breakdown), prior to the addition of agonists or antagonists. When present, antagonists were incubated for 20 min at 23°C prior to stimulation with agonist; concentrations ranged from 0.00003 to 3 µmol·L−1 for orexin-A and 0.0001 to 10 µmol·L−1 for orexin-B. After 45 min incubation at 37°C with agonist, the assay was terminated by the aspiration of the assay buffer and the addition of 100 µL 20 mmol·L−1 formic acid to the cells. After shaking for 30 min at 23°C, a 40 µL aliquot was mixed with 80 µL of yttrium silicate beads (12.5 mg·mL−1) that bind to the IP (but not inositol) and shaken for 30 min at 23°C. Assay plates were centrifuged for 2 min at 750×g prior to counting on a Packard Top-count microplate scintillation counter with quenching correction (PerkinElmer Life and Analytical Sciences).

    Intracellular Ca2+ mobilization assay

    The CHO(dHFr-)-hOX1 and -hOX2 stable cells lines were seeded at 5 × 104 cells per well in the poly-D-lysine treated, 96-well, black/clear-bottomed plates. Twenty-four hous later, cells were loaded for 1 h at 37°C with 4 µmol·L−1 Flou-4 acetoxymethyl ester in FLIPR buffer (1 × HBSS, 20 mmol·L−1 HEPES, 2.5 mmol·L−1 Probenecid). Cells were washed 5 times with FLIPR buffer to remove excess dye and intracellular calcium mobilization; [Ca2+]i were measured using a Fluorometric Imaging Plate Reader (FLIPR-96, Molecular Devices, Menlo Park, CA) as described previously (Malherbe et al., 2006). Orexin-A or orexin-B (50 mmol·L−1 stock solution in DMSO) were diluted in FLIPR buffer plus 0.1% BSA. The EC50 and EC80 values of orexin were measured daily from standard agonist concentration-response curves in CHO(dHFr-)-hOX1 or -OX2 receptor stable cell line. All compounds were dissolved in 100% DMSO, and diluted in FLIPR buffer to a 5× stock (2.5% DMSO). This stock was then applied to the cells at a final DMSO concentration of 0.5%. Inhibition curves were determined by addition of 11 concentrations (0.0001–10 µmol·L−1 in FILPR buffer) of inhibitory compounds and using EC80 value of orexin-A or orexin-B as agonist (a concentration which gave 80% of maximum agonist response, determined daily). The antagonists were applied 25 min (incubation at 37°C) before the application of the agonist. Responses were measured as peak increase in fluorescence minus basal, normalized to the maximal stimulatory effect induced by EC80 value of orexin-A or orexin-B. Inhibition curves were fitted according to the Hill equation: y = 100/(1+(x/IC50)nH), where nH= slope factor using Prism 4.0 (GraphPad software). Kb values were calculated according to the following equation Kb= IC50/(1 +[A]/EC50), where A is the concentration of agonist added that is very close to agonist EC80 value, and IC50 and EC50 values were derived from the antagonist inhibition and orexin agonist curves respectively.

    Radioligand binding to tissue sections and quantitative receptor autoradiography

    All animal care and experimental procedures were approved by the City of Basel Cantonal Animal Protection Committee, based on adherence to Federal and local regulations. Mice and rats were housed in separate holding rooms at controlled temperature (20–22°C) and 12 h light/dark cycle (lights on 06:00 h). All animals were allowed ad libitum access to food and water.

    Male CD Sprague-Dawley rats weighing 150–180 g were killed by decapitation; brains were rapidly dissected and immediately frozen in dry ice. Coronal cryostat-cut sections (10 µm thick) were mounted on Histobond glass slides (Marienfeld Laboratories Glassware, Germany) dried at room temperature and stored at −20°C. Sections were pre-incubated 2 × 10 min in assay buffer (1 mmol·L−1 CaCl2, 5 mmol·L−1 MgCl2, 25 mmol·L−1 HEPES, pH 7.4) and then for 60 min in assay buffer containing 1 nmol·L−1[3H]EMPA (all incubations at room temperature). Sections were then rinsed in 2 × 5 min in ice-cold assay buffer (2 × 5 min). This was followed by three rapid washes in distilled water at 4°C. NSB was determined in the presence of 10 µmol·L−1 Cp-5 or other orexin receptor antagonists.

    Brain sections were exposed, together with tritium microscales, to tritium-sensitive imaging plates (BAS-TR2025) for 5 days. Plates were scanned in a Fujifilm BAS-5000 high resolution phosphor imager and images quantified with an MCID M2 image analysis system (Imaging Research Inc., St. Catherines, Ontario, Canada). Sections were then stained with cresyl violet, photographed and pictures obtained were compared with a parent autoradiogram to allow unambiguous identification of the regions displaying binding of [3H]EMPA.

    Determination of OX2 receptor occupancy using ex vivo [3H]EMPA autoradiography

    Male CD Sprague-Dawley rats were given vehicle (1% Tween-80 in physiological saline) or almorexant (3, 10 or 30 mg·kg−1) (i.p., n= 2 per group). Thirty minutes after dosing, animals were sacrificed by decapitation; brains were rapidly dissected and immediately frozen in dry ice. Cryostat coronal sections were processed for [3H]EMPA receptor autoradiography as described above.

    Pharmacokinetics of EMPA in mice and rats

    Pharmacokinetic experiments were performed in male NMRI mice and Wistar rats. Mice were dosed either i.v. (into the tail vain) or p.o. (microsuspension, as a gavage). At defined time points, terminal plasma and brain tissue was collected. Two mice per group were killed at 0.083, 0.333, 1, 2, 4 and 7 h after the i.v. administration of 10.77 mg·kg−1 EMPA or 0.25, 0.5, 1, 2, 4 and 7 h after the p.o. administration of 18.04 mg·kg−1 EMPA. Rats were given a single oral dose (19.71 mg·kg−1, microsuspension, as a gavage) or i.v. (11.79 mg·kg−1, via a jugular vein). Plasma and brain samples were collected after killing from two rats per group at 0.083, 0.25, 0.5, 1, 2, 4 and 8 h (i.v.) or 0.25, 0.5, 1 and 2 h (p.o.) after dosing. Concentrations of EMPA were determined using quantitative liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). Pharmacokinetic parameters were calculated by non-compartmental analysis of plasma concentration-time curves using WinNonlin, version 4.1 software (Pharsight Corporation, Mountain View, CA).

    In vivo evaluation of EMPA

    Animals and drug treatment

    Male NMRI mice (20–30 g) supplied from Iffa Credo, Lyon, France and Male Wistar rats (196–237 g) supplied from RCC Ltd., Fullinsdorf, Switzerland were used. EMPA was prepared immediately prior to use in 0.3% (w/v) Tween-80 in physiological saline (0.9% NaCl) and injected i.p. at a volume of 10 mL·kg−1 body weight for mice and 5 mL·kg−1 for rats. All doses are expressed as that of the base.

    Reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion in mice

    A computerized Digiscan 16-Animal Activity Monitoring System (Omnitech Electronics, Colombus, OH) was used to quantify locomotor activity (LMA). Data were obtained simultaneously from eight Digiscan activity chambers placed in a soundproof room with a 12 h light/dark cycle. All tests were performed during the light phase (6 am to 6 pm). Each activity monitor consisted of a Plexiglas box (20 × 20 × 30.5 cm) with sawdust bedding on the floor surrounded by invisible horizontal and vertical infrared sensor beams. Cages were connected to a Digiscan Analyzer linked to a PC that constantly collected the beam status information. The activity detector operates by counting the number of times the beams change from uninterrupted to interrupted or vice versa. Records of photocell beam interruptions, for individual animals, typically were taken every 5 min over the duration of the test session. Mice were first transferred from home cages to recording chambers for a 50 min habituation phase during which they were allowed to freely explore the new environment. Mice were then injected i.p. with EMPA (1, 3, 10, 30, 100, 300 mg·kg−1, n= 8 mice per dose). Ten minutes later, mice were briefly anaesthetized with isoflurane inhalation to allow i.c.v. injection of 5 µL of either artificial cerebrospinal fluid (CSF) or [Ala11,D-Leu15]orexin-B at a dose of 3 µg. Mice were then immediately replaced in the test compartments and LMA was recorded during the following 30 min.

    Spontaneous locomotor activity in rats during the dark (active) phase

    Male Wistar rats (∼150 g at arrival) housed four per cage (Makrolon cages 1800 cm2) with free access to food and water were allowed 2 weeks of acclimatization to a reversed light/dark animal room (dark cycle: 10.00 am to 10.00 pm) prior to testing. On test days, LMA was monitored by a computerized Digiscan Animal Activity Monitoring system as described above (Omnitech Electronics, Columbus, OH). The activity monitoring chambers were made of Plexiglas (41 × 41 × 30 cm W × L × H) and contained a thin layer of sawdust bedding. One rat per cage was monitored at the same time. One hour after the dark period onset, rats were injected i.p. with EMPA (3, 10, 30 mg·kg−1, n= 8 rats per dose) and immediately placed into the activity monitoring chambers. LMA was then recorded in 5 min time bins for a period of 30 min.

    Motor coordination and balance in rats

    Male Wistar rats (∼200 g body weight) were trained to remain on a horizontal metal rod (rotarod, Ugo Basile, Biological Research Apparatus, Varese, Italy) rotating at a fixed speed until criterion level (120 s on rod) was reached. The rotarod was 7 cm wide, 5 cm in diameter and 25 cm above the bench. The following day, animals were injected i.p. with vehicle or EMPA (3, 10 or 30 mg·kg−1; n= 8 per group). Animals were tested for rotarod performance at 8 r.p.m. and then at 16 r.p.m. (total time spent on the rod, maximum 120 s) 10 min after injection. Rats were allowed a maximum of three trials to remain on the rotarod for 120 s; assessment terminated when the animal fell from the rotarod or reached criterion level. The mean time over the number of trials completed per rat was calculated.


    All parameters were analysed with a repeated measure anova, followed in significant cases by a Dunnett's t-test. A P-value of 0.05 was accepted as statistically significant.


    EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulphonyl)-amino]-N-pyridin-3-ylmethyl-acetamide, WO2004033418A2), almorexant (ACT-078573, (R)-2-{(S)-6,7-dimethoxy-1-[2-(4-trifluoromethyl-phenyl)-ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide, WO2005118548-A1) (Brisbare-Roch et al., 2007), Cp-1 ((R)-2-{(S)-6,7-dimethoxy-1-[2-(6-trifluoromethyl-pyridin-3-yl)-ethyl]-3,4-dihydro-1H-isoquinolin-2-yl}-N-methyl-2-phenyl-acetamide, WO2005118548-A1), Cp-2 (1-(9-oxo-8-trifluoromethyl-1,2,3,9-tetrahydro-pyrrolo[2,1-b]quinazolin-3-yl)-1-((S)-1-phenyl-ethyl)-3-(2-trifluoromethoxy-phenyl)-urea, WO2004004733A1), Cp-3 (2-methyl-5-phenyl-thiazole-4-carboxylic acid cyclobutyl-[3-(4-fluoro-phenoxy)-propyl]-amide, WO2006110626A1), Cp-4 (2-[[4-chloro-2-(hydroxy-phenyl-methyl)-phenyl]-(3,4-dimethoxybenzenesulphonyl)-amino]-N-methyl-acetamide, WO2006024779-A1), Cp-5 ((S)-1-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-3,3-dimethyl-2-[(pyridin-4-ylmethyl)-amino]-butan-1-one, WO2001085693-A1) (Hirose et al., 2003), and SB 674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone) (Langmead et al., 2004) were synthesized in the chemistry department of F. Hoffmann-La Roche according to procedures described in patent literature. [3H]EMPA (specific activity: 94.3 Ci mmol−1) and [3H]SB 674042 (specific activity: 24.4 Ci mmol−1) were synthesized by Drs Philipp Huguenin and Thomas Hartung at the Roche chemical and isotope laboratories, Basel, Switzerland. SB 334867 (N-(2-methyl-6-benzoxazolyl)-N’’-1,5-naphthyridin-4-yl urea) (Tocris 1960), orexin-A (Tocris 1455), orexin-B (Tocris 1456) and [Ala11,D-Leu15]orexin-B (Tocris 2142) were purchased from Tocris Bioscience (Bristol, UK). [myo-1,2-3H]inositol with PT6-271 (TRK911, specific activity: 16.0 Ci mmol−1; GE Healthcare, Chalfont St. Giles, UK) and yttrium silicate RNA binding beads (RPNQ0013) were purchased from GE Healthcare.


    Characterization of [3H]EMPA binding and displacement studies

    EMPA was previously described in the patent WO2004033418A2 to be a selective OX2 receptor antagonist. For the current study, EMPA was tritiated ([3H]EMPA, Figure 1). To characterize the in vitro binding of [3H]EMPA, saturation binding analysis was performed at binding equilibrium (1 h incubation at 23°C), on membranes isolated from the HEK293 transiently transfected with the human and rat OX2 receptors. The saturation isotherm and Scatchard plot of [3H]EMPA binding to human OX2-HEK293 cell membranes are shown in Figure 2A. The saturation isotherm was monophasic ([3H]EMPA concentrations 0.01–12 nmol·L−1) and best fitted to a one-site model. Similarly, the Scatchard plot was linear (see inset of Figure 2A).

    Details are in the caption following the image

    Chemical structures of the selective OX1, OX2 and dual OX1/OX2 receptor antagonists. T, tritium.

    Details are in the caption following the image

    Binding characteristic of [3H]EMPA to membrane preparations from HEK293 cells transiently expressing hOX2 receptors. (A) Saturation binding curve and Scatchard plot (inset) of [3H]EMPA binding to membranes from HEK293 cells transfected transiently with hOX2 receptors. Specific binding (SB) was obtained by calculating the difference between total binding (TB) and non-specific binding (NSB), measured in the presence of 10 µmol·L−1 EMPA. Each data point is ±SEM (bars) of three individual experiments performed in triplicate. The data were analysed by non-linear regression analysis using GraphPad Prism 4.0 software and a single-site binding model. Time course for the association (B) and dissociation (C) of [3H]EMPA binding to hOX2 membranes. Each data point is ±SEM (bars) of three individual experiments, with four replicates.

    Binding kinetics of [3H]EMPA to membrane preparations from HEK293 cells transiently expressing hOX2 receptors are shown in Figure 2B and C and the kinetic parameters in Table 1. The association binding of [3H]EMPA to the hOX2 receptors was rapid with half-maximal binding occurring at 6.3 min and reaching equilibrium within 20 min. The data were fitted by a one-phase exponential model with the association rate constant of 0.028 ± 0.06 nmol·L−1·min−1. The dissociation rate for [3H]EMPA binding to the hOX2 receptors was determined by the addition of an excess amount of EMPA after equilibrium was reached. The reversal of binding for EMPA was complete with t1/2 value of 9 min. The calculations of the apparent KD value derived from the kinetic experiments was 2.74 ± 0.20, which was higher than that of equilibrium KD value of 1.1 ± 0.1 nmol·L−1.

    Table 1. Kinetic parameters for the association and dissociation of [3H]EMPA in membrane preparations from HEK293 cells transiently expressing hOX2 receptors
    Compound Association kinetic Dissociation kinetic Apparent KD nmol·L-1
    Kob min - 1 Kon nmol·L - 1·min - 1 t1/2 min Koff min - 1 t1/2 min
    [3H]EMPA 0.11 ± 0.01 0.028 ± 0.06 6.29 ± 0.47 0.078 ± 0.00 8.89 ± 0.25 2.74 ± 0.20
    • The Kob (observed on rate), Koff (observed off rate), Kon (calculated on rate), t1/2 (half-maximal binding) and KD (apparent dissociation constant) values are ±SEM, calculated from three independent experiments (each performed with four replicates) as described under Methods.

    [3H]SB 674042, an OX1 receptor selective radioligand antagonist, binding to hOX1 membrane was previously described (Langmead et al., 2004). Hence, the selectivity of EMPA for OX2 versus OX1 receptors was determined using [3H]SB 674042 and [3H]EMPA competition binding assays to the membranes isolated from the HEK293 transiently transfected with the hOX1 and hOX2 receptors. As seen in Figure 3A, [3H]SB 674042 was weakly displaced by EMPA from hOX1 membrane with IC50= 1900.0 ± 157.0 nmol·L−1, Ki= 900.0 ± 67.0 nmol·L−1, nH= 1.0 ± 0.0. While [3H]EMPA was strongly displaced by EMPA from hOX2 membrane with IC50= 2.3 ± 0.5 nmol·L−1, Ki= 1.1 ± 0.2 nmol·L−1, nH= 1.0 ± 0.0. Furthermore, to assess the pharmacological profile of [3H]EMPA in competition binding assay, selective OX1, OX2 and dual OX1/OX2 receptor antagonists that had been previously described in the patent literature were synthesized: the selective OX1 receptor antagonists SB 334867 (Smart et al., 2001) and SB 674042 (Langmead et al., 2004); selective OX2 receptor antagonists Cp-4 and Cp-5 (Hirose et al., 2003) and dual OX1/OX2 receptor antagonists almorexant (Brisbare-Roch et al., 2007), Cp-1, Cp-2 and Cp-3 are shown in Figure 1. Potencies of these antagonists in inhibition of [3H]EMPA binding to hOX2-HEK293 cell membranes are shown in Figure 3B with Ki and nH values in Table 2.

    Details are in the caption following the image

    (A) The displacement of [3H]SB 674042 and [3H]EMPA binding by EMPA in membrane preparations from HEK293 cells transiently expressing hOX1 and hOX2 receptors respectively. (B) The displacement of [3H]EMPA binding by OX1-selective, OX2-selective and dual OX1/OX2 receptor antagonists in the hOX2 cell membrane. The [3H]SB 674042 (0.7 nmol·L−1) and [3H]EMPA (1.1 nmol·L−1) were used at a concentration equal to their KD values in these competition binding experiments. Each data point is ±SEM (bars) of three individual experiments performed in duplicate.

    Table 2. [3H]EMPA displacement by various selective OX1, OX2 and dual OX1/OX2 receptor antagonists in the membrane preparations from HEK293 cells transiently expressing hOX2 receptors
    OX antagonists Ki nmol·L - 1 nH
    EMPA 1.10 ± 0.24 1.01 ± 0.01
    Almorexant 4.35 ± 0.27 1.22 ± 0.05
    Cp-1 2.94 ± 0.66 1.27 ± 0.04
    Cp-2 6.46 ± 0.60 1.18 ± 0.04
    Cp-3 7.76 ± 0.50 0.87 ± 0.08
    Cp-4 5.98 ± 0.51 1.10 ± 0.04
    Cp-5 51.10 ± 3.44 1.02 ± 0.09
    SB 334867 >10 000
    SB 674042 334.0 ± 110.0 1.0 ± 0.2
    • Ki and Hill slope (nH) values for [3H]EMPA binding inhibition by various antagonists were calculated as described under Methods. Values are ±SEM of the Ki calculated from three independent experiments, each performed in duplicate.

    Of note is the use of two cell systems for binding (HEK293 cells transiently transfected with hOX2 receptors) and functional studies [FLIPR and IP accumulation assays using CHO(dHFr-)-hOX2 receptor stable cell in the current study]. As HEK293 cells were adapted to grow and be transiently transfected in suspension in spinner flasks, it was possible to produce and prepare large quantities of transfected cells and membranes required for binding studies. Experiments with membranes prepared from HEK293-hOX2 and CHO(dHFr-)-hOX2 cell systems showed similar KD values (1.1 vs. 0.7 nmol·L−1) for [3H]EMPA binding on these membranes; the only difference between these two cell systems were Bmax values (38 vs. 2.4 pmol·mg−1 protein) and per cent non-specific/total binding (NSB/TB% of 1.3% vs. 11% respectively) that indicated a higher level of expression and lower NSB/TB% in HEK293 cells than that of CHO cells. However, since [3H]EMPA binds to a single site in a saturable manner, the expression level of the receptor does not influence the determination of KD. Hence, HEK293 cells were used for binding studies.

    Antagonist potency and the inhibition mode of EMPA

    In CHO(dHFr-) cells stably expressing hOX2 receptors, EMPA inhibited orexin-A- or orexin-B-evoked [Ca2+]i response with IC50= 8.8 ± 1.7 nmol·L−1, nH= 0.9 ± 0.0 and IC50= 7.9 ± 1.7 nmol·L−1, nH= 1.0 ± 0.1 respectively (Figure 4). EMPA-mediated inhibition of orexin-A-induced [Ca2+]i response was used to address further the selectivity of EMPA for hOX2 over hOX1 receptors in the functional assay. As seen in Figure 4, orexin-A-evoked [Ca2+]i response was poorly inhibited by EMPA with IC50 > 10 000 nmol·L−1 in the CHO(dHFr-)-hOX1 stable cells.

    Details are in the caption following the image

    Inhibition of orexin induced Ca2+ mobilization by EMPA at hOX1- and hOX2-CHO-dHFr- stable cell lines. Concentration-dependent inhibition orexin-A and orexin-B stimulated increases in [Ca2+]i by EMPA as assayed using the Ca2+-sensitive dye, Flou-4 and a Fluorometric Imaging Plate Reader (FLIPR-96). Each curve represents ±SEM (bars) of the three dose-response measurements (each performed in duplicate).

    To characterize the inhibition mode of EMPA, the concentration-response curves for [3H]IP formation stimulated by orexin-A or orexin-B have been measured in the presence of various concentrations (0, 3, 10, 30, 100, 300 and 600 nmol·L−1) of EMPA in the CHO(dHFr-)-hOX2 stable cell line. As seen in Figure 5A and C, orexin-A (0.03 nmol·L−1–3 µmol·L−1) and orexin-B (0.1 nmol·L−1–10 µmol·L−1) elicited concentration-dependent increases in the accumulation of [3H]IP in the hOX2 receptor expressing cells with the EC50, nH values of 1.1 ± 0.1 nmol·L−1, 1.4 ± 0.1 and 2.4 ± 0.9 nmol·L−1, 0.6 ± 0.1 respectively. EMPA behaved as a competitive antagonist at hOX2 receptors, shifting both orexin-A and orexin-B concentration-response curves to the right without changing their maximal responses (Figure 5A and C). The apparent antagonist potency (pA2) and the Schild slope values calculated from orexin-A and orexin-B Schild analyses (Figure 5B and D) and are given in Table 3. As seen in Table 3, the functional potency of EMPA derived from FLIPR assay (pKb) was in good agreement with that of the [3H]IP accumulation assay (pA2).

    Details are in the caption following the image

    Schild analyses showing the competitive mode of antagonism by EMPA at OX2 receptors. Concentration-response curves for [3H]IP formation stimulated by orexin-A (A) and orexin-B (C) in the absence and presence of various concentrations of EMPA in CHO(dHFr-)-hOX2 stable cell line. Schild plots for antagonism by EMPA (B and D). The EC50 and EC50’ values, which derived from orexin-A and orexin-B concentration-response curves in the absence and presence of increasing fixed concentrations of EMPA (A and C), were used to calculate the dose ratios (DR = EC50’/EC50) and plotted according to Schild regression in panels b and d. Each concentration-response curve is ±SEM (bars) of three individual experiments, each performed with four replicates.

    Table 3. Antagonism profile of EMPA as analysed in FLIPR and Schild plot
    [Ca2 + ]i mobilization Schild analysis
    pKb Kb nmol·L-1 nH pA2 Kba nmol·L - 1 Schild slope
    Orexin-A 9.1 0.8 ± 0.2 0.9 ± 0.0 8.6 2.8 1.0
    Orexin-B 9.2 0.6 ± 0.0 1.0 ± 0.1 8.8 1.6 0.9
    • pKb, Kb and Hill coefficient (nH) values for the inhibition of orexin-A- or orexin-B-evoked [Ca2+]i response in the CHO(dHFr-)-hOX2 receptor stable cell line were calculated as described under Methods. Data are ±SEM of the three dose-response measurements (each performed in duplicate). Schild constants for antagonism of orexin-A- or orexin-B-induced accumulation of [3H]IP by EMPA in the CHO(dHFr-)-hOX2 cells. The apparent antagonist potency (pA2) and Schild slope values of EMPA were determined from Schild plot analyses shown in Figure 5B and D.

    Selectivity profile of EMPA

    The pharmacological specificity of EMPA was confirmed by assessment in radioligand binding assays in a broad CEREP screen (Paris, France) (http://www.cerep.fr) (Table 4). Among the 80 receptors in the CEREP broad screen, 30 were peptide receptors. For the selection of peptide receptors in this broad screen, the amino acids forming the transmembrane domains (7TMD) of human OX2 receptors were aligned with those of the large number of peptide receptors in the protein database (Swissprot). The hOX2 receptor 7TMD displayed a similarity of 37%, 29% and 23% to the 7TMD of hNPY2 (neuropeptide Y2), hCCK1 (cholesystokinin 1) and hNK2 (neurokinin 2) receptors, respectively, and these classes of GPCRs were therefore included in our selectivity screen. EMPA was inactive (<50% activity at 10 µmol·L−1) at all targets tested with the exception of the hV1a (vasopressin) and KOP (κ opiate) receptors, where it caused 79% and 65%, respectively, displacement of specific binding at 10 µmol·L−1. However, subsequent concentration-response curves with EMPA showed an IC50= 5.75 µmol·L−1, Ki= 2.63 µmol·L−1, nH= 1.0 and IC50= 12.8 µmol·L−1, Ki= 5.8 µmol·L−1, nH= 1.0 in the binding assay at human and mouse V1a receptors respectively. EMPA also had Ki= 11.3 µmol·L−1, nH= 1.4 in the binding assay at hKOP. Hence, EMPA had negligible binding affinities at the human and mouse V1a and hKOP receptors.

    Table 4. CEREP selectivity screen in the broad radioligand binding assays were undertaken to determine the pharmacological activity of EMPA
    Target Reference compound % control (10 µmol·L - 1) mean SB Target Reference compound % control (10 µmol·L - 1) mean SB
    A1 (h) DPCPX 82 M4 (h) 4-DAMP 107
    A2A (h) NECA 95 M5 (h) 4-DAMP 98
    A3 (h) IB-MECA 106 NK1 (h) [Sar9,Met(O2)11]-SP 109
    α1 (non-selective) Prazosin 102 NK2 (h) [Nle10]-NKA(4-10) 93
    α2 (non-selective) Yohimbine 92 NK3 (h) SB 222200 82
    β1 (h) Atenolol 97 Y1 (h) NPY 110
    β2 (h) ICI 118551 99 Y2 (h) NPY 97
    AT1 (h) Saralasin 101 NT1 (h) (NTS1) Neurotensin 98
    AT2 (h) Saralasin 104 Delta 2 (h) (DOP) DPDPE 105
    BZD (central) Diazepam 84 Kappa (KOP) U 50488 30
    BZD (peripheral) PK 11195 70 mu (h) (MOP) (agonist site) DAMGO 91
    BB (non-selective) Bombesin 77 ORL1 (h) (NOP) Nociceptin 115
    B2 (h) NPC 567 93 PACAP (h) (PAC1) PACAP1-38 101
    CGRP (h) hCGRPalpha 132 PCP MK 801 101
    CB1 (h) CP 55940 94 TXA2/PGH2 (h) (TP) U 44069 73
    CCKA (h) (CCK1) CCK-8 97 P2X Alpha, beta-MeATP 110
    CCKB (h) (CCK2) CCK-8 95 P2Y dATPalpha S 89
    D1 (h) SCH 23390 99 5-HT1A (h) 8-OH-DPAT 98
    D2S (h) (+)butaclamol 95 5-HT1B (h) Serotonin 95
    D3 (h) (+)butaclamol 101 5-HT2A (h) Ketanserin 89
    D4.4 (h) Clozapine 95 5-HT2C (h) RS-102221 81
    D5 (h) SCH 23390 97 5-HT3 (h) MDL 72222 103
    ETA (h) Endothelin-1 99 5-HT5A (h) Serotonin 100
    ETB (h) Endothelin-3 95 5-HT6 (h) Serotonin 92
    GABA (non-selective) GABA 107 5-HT7 (h) Serotonin 98
    GAL1 (h) Galanin 96 Sigma (non-selective) Haloperidol 106
    GAL2 (h) Galanin 100 sst (non-selective) Somatostatin-14 97
    PDGF PDGF BB 94 VIP1 (h) (VPAC1) VIP 104
    CXCR2 (h) (IL-8B) IL-8 101 V1a (h) [d(CH2)51,Tyr(Me)2]-AVP 21
    TNF-alpha (h) TNF-alpha 93 Ca2+ channel (L, verapamil site) (phenylalkylamines) D 600 108
    CCR1 (h) MIP-1alpha 101
    H1 (h) Pyrilamine 106 K + V channel Alpha-dendrotoxin 98
    H2 (h) Cimetidine 93 SK + Ca channel Apamin 89
    MC4 (h) NDP-alpha-MSH 103 Na+ channel (site 2) Veratridine 100
    MT1 (h) Melatonin 59 Cl channel Picrotoxinin 94
    M1 (h) Pirenzepine 83 NE transporter (h) Protriptyline 106
    M2 (h) Methoctramine 93 DA transporter (h) BTCP 71
    M3 (h) 4-DAMP 95 5-HT transporter (h) Imipramine 108

    [3H]EMPA binding to rat brain sections

    The distribution and abundance of in vitro binding sites of [3H]EMPA was investigated in coronal rat brain sections using autoradiography and image analysis (Figure 6). A high density of specific binding was observed in many brain regions including the limbic cortices, hippocampus, striatum and hypothalamic nuclei. NSB, determined in the presence of 10 µmol·L−1 Cp-5, a selective OX2 receptor antagonist (Figure 6B) was <8% of TB.

    Details are in the caption following the image

    Regional distribution of [3H]EMPA binding sites in coronal sections of rat brain revealed by autoradiography. Panel A, C and D show total binding (1 nmol·L−1[3H]EMPA); B shows non-specific binding (+10 µmol·L−1 Cp-5); (E) Regional abundance of [3H]EMPA specific binding (1 nmol·L−1) measured by quantitative autoradiography and image analysis. Mean values are expressed as fmol mg−1 protein. Competition with 10 µmol·L−1 Cp-5, no binding is evident (panel B). Abbreviations used in the figure: AcbSh, nucleus accumbens shell; IG, indusium griseum; CA3 region of the hippocampus; MD mediodorsal thalamic nuclei; DR, dorsal raphe nucleus; S, subiculum; RtTg, reticulotegmental neuclei of the pons.

    Ex vivo receptor occupancy studies using [3H]EMPA autoradiography

    To further characterize the specificity of [3H]EMPA binding in the rat brain, ex vivo binding experiment were conducted to investigate the ability of the known orexin antagonist almorexant to block the binding of [3H]EMPA. Administration of almorexant (3, 10 or 30 mg·kg−1, i.p.) resulted in a dose-dependent decrease of the binding of [3H]EMPA to the cortical layer 6 (Figure 7A), hilus dentate gyrus (Figure 7B) and all other brain regions evaluated. These data provide a first estimation of OX2 receptor occupancy produced by almorexant after systemic administration.

    Details are in the caption following the image

    OX2 receptor occupancy by almorexant (3, 10, 30 mg·kg−1 i.p.) in the rat cortical layer 6 (A) and hilus dentate gyrus (B) determined using ex vivo[3H]EMPA autoradiography.

    Physicochemical and pharmacokinetics properties of EMPA in mice and rats

    The physicochemical properties of EMPA are: MW = 454.4 g·mol−1; lipophilicity clogP/logD of 3.43/2.3 at pH 7.4; permeation coefficient Pe= 5.5 × 10−6 cm·s−1 (Parallel Artificial Membrane Permeation Assay, PAMPA); thermodynamic solubility of 278 µg·mL−1; pKa of 4.62 and a polar surface area of 73 Å2. With the exception of 3A4, EMPA does not inhibit major cytochrome P450 isoenzymes (human liver microsomes IC50 values: 3A4: 0.7 µmol·L−1; 2D6 and 2C9: >50 µmol·L−1). EMPA displayed a weak activity as a substrate for P-glycoprotein mediated efflux transport. Furthermore, EMPA inhibited human ERG channel with an IC50 value of 3.6 µmol·L−1 (27% at 1 µmol·L−1, 69% at 10 µmol·L−1).

    The oral bioavailability and pharmacokinetics of EMPA were evaluated in male NMRI mice and Wistar rats. The mean pharmacokinetic parameters of EMPA after single intravenous (i.v.) or oral (p.o.) bolus administration in mice and rat are given in Table 5. The concentration of EMPA in rat brain measured at 2 and 4 h was <10 ng·mL−1. EMPA is highly protein bound (2.8%, 4.7% and 10% free fraction in human, mouse and rat plasma respectively). The stability of EMPA measured 1 h/4 h in human, mouse and rat plasma was 133%/96%, 105%/109% and 136%/135% respectively.

    Table 5. Pharmacokinetic assessment of EMPA after i.v. and p.o. administration to mice and rats
    Route Mouse Rat
    Plasma Brain Plasma
    i.v. p.o. i.v. p.o. i.v. p.o.
    Dose, mg·kg−1 10.77 18.04 10.77 18.04 11.79 19.71
    Cmax/dose, ng·mL−1 512.1 68.2 266.2 13.7 421.9 1.8
    Tmax, h 0 0.5 0 0.25 0 0.63
    AUC/dose, ng·h·mL−1 201.4 43.7 56.3 7.3 127.4 1.4
    T1/2, h 1.85 0.98 0.15 0.14 0.81 0.37
    Vss, L·kg−1 1.31 2.71 2.07
    CL, mL·min−1·kg−1 82.8 295.8 131.7
    F, % 21.7 1.1
    Fu, % 4.7 10.0
    • Cmax, maximum concentration; Tmax, time at which maximum concentration was observed; AUC, area under the plasma concentration versus time curve; CL, clearance; Vss, volume of distribution at steady state; T1/2, terminal half-life; F, bioavailability; Fu, fraction unbound. Values are means for mice (n= 2 per time point) and for rats (n= 2).

    Monitoring of EMPA exposure in mice and rats at the completion of the behavioural procedures

    To determine EMPA exposure, plasma and brain exposure of EMPA were monitored at the end of the behavioural procedures mentioned below in mice and rats. In the reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion test in mice, the plasma concentrations, measured 45 min after i.p. injection of EMPA at doses 30, 100 and 300 mg·kg−1 were 73, 2182 and 23 300 ng·mL−1 respectively. The brain level of EMPA, given at a dose of 300 mg·kg−1 and measured 45 min after i.p. injection was 16 419 ng·mL−1. In the rat LMA test, the plasma levels of EMPA, given at doses of 3, 10 and 30 mg·kg−1and determined 65 min after i.p. injection were 7, 32 and 68 ng·mL−1 respectively. However, the brain level of EMPA (30 mg·kg−1) in rats, measured 65 min after i.p. dosing, was approximately 8 ng·mL−1.

    In vivo activity of EMPA

    Reversal of [Ala11,D-Leu15]orexin-B-induced hyperlocomotion in mice

    As shown on Figure 8, and as compared with animals injected with artificial CSF (open symbol), mice given 3 µg of the preferential OX2 receptor agonist [Ala11,D-Leu15]orexin-B i.c.v. exhibited marked increases in LMA (>300%). EMPA (1, 3, 10, 30, 100, 300 mg·kg−1 i.p.) dose-dependently reversed this [Ala11,D-Leu15]orexin-B-induced hyperlocomotion [F(6,49) = 15.1, P < 0.001] without itself significantly affecting LMA [F(6,41) = 1.12, P > 0.05].

    Details are in the caption following the image

    Effects of EMPA (0, 1, 3, 10, 30, 100, 300 mg·kg−1 i.p.) on [Ala11,D-Leu15]orexin-B-induced hyperlocomotion (orexin + EMPA) and spontaneous locomotor activity (CSF + EMPA) in mice. Data points indicate mean horizontal activity counts per group, error bars indicate SEM (n= 8 per group). # indicates significant difference from vehicle/CSF group, and asterisks indicate significant difference from orexin/vehicle group in post hoc testing (P < 0.05 at least).

    Spontaneous locomotor activity in rats during the dark (active) phase

    When placed in the activity monitoring chambers, vehicle-treated animals initially exhibited a marked hyperactivity that rapidly adapted over the 30 min recording period. In drug-treated animals, EMPA (3, 10, 30 mg·kg−1 i.p.) induced a significant and dose-dependent reduction in the baseline LMA [F(3,28) = 5.24, P < 0.01; Figure 9]. This decrease in locomotion was already evident 5 min after giving the 10 and 30 mg·kg−1 doses. When activity was summated over a 10 min period (the inset in top-right corner of Figure 9), EMPA demonstrated a clear dose-dependent inhibition of spontaneous activity as compared with vehicle-treated animals [F(3,28) = 4.18, P < 0.05].

    Details are in the caption following the image

    Effects of EMPA (3, 10, 30 mg·kg−1 i.p.) on spontaneous locomotor activity in rat. Each data point indicates mean horizontal activity counts per 5 min time period; error bars indicate SEM (n= 8 per group). The bar chart in the inset depicts the dose-dependent effect of the compound on cumulative horizontal activity over the time period 10–15 min. Asterisks indicate significant difference from vehicle-treated group in post hoc testing (P < 0.05 at least).

    Motor coordination and balance in rats

    In the rotarod test, no significant motor disturbances were observed following treatment with EMPA (3, 10 and 30 mg·kg−1 i.p.) when animals were examined on a bar rotating at 8 r.p.m. [F(3,28) = 0.71, P > 0.05] or 16 r.p.m. [F(3,28) = 0.59, P > 0.05], as shown in Figure 10.

    Details are in the caption following the image

    Rotarod performance following injection of 3, 10 or 30 mg·kg−1 i.p. of EMPA compared to vehicle controls. Bars indicate mean time spent on rotarod in seconds (maximum 120 s) ± SEM.


    The orexin system and OX2 receptors may play a role in stress and in the regulation of emotional responses via its interaction with the corticotropin releasing factor (CRF) system in the hypothalamus (Sakamoto et al., 2004). I.c.v. injection of orexin-A induces grooming (stress response) that is blocked in part by a CRF antagonist (Ida et al., 2000). OX2 receptors are predominantly expressed in the paraventricular nucleus in the hypothalamus and orexin neurons projecting to CRF neurons express mainly OX2 receptors (Winsky-Sommerer et al., 2004; 2005). Therefore, OX2 receptor stimulation activates the hypothalamo-pituitary-adrenal axis. Interestingly, a recent study has also shown that a selective OX2 receptor antagonist, N-{(1S)-1-(6,7-dimethoxy-3,4-dihydro-2(1H)-isoquinolinyl)carbonyl}-2,2-dimethylpropyl)-N-amine, was able to attenuate the orexin-A-induced increases in plasma ACTH (Chang et al., 2007). Thus far, however, there are few reports describing biochemical and pharmacological characterization of selective OX2 receptor antagonists. Here, we describe the in vitro and in vivo properties of a highly selective OX2 receptor antagonist, EMPA.

    [3H]EMPA binds to a single saturable site on recombinantly expressed human and rat OX2 receptors (Bmax of 38.29 ± 0.50 and 6.62 ± 0.60 pmol·mg−1 protein respectively) with high affinity (KD values of 1.11 ± 0.05 nmol·L−1 and 1.36 ± 0.04 nmol·L−1 respectively). Similarly, EMPA was able to displace the [3H]EMPA binding from cell membranes containing human and rat OX2 receptors, with Ki values of 1.10 ± 0.24 nmol·L−1 and 1.45 ± 0.13 nmol·L−1 and Hill values of 1.01 ± 0.01 and 0.93 ± 0.08 respectively. At the KD value, NSB for [3H]EMPA was approximately 1.3% of total bound radioactivity. EMPA displayed a high selectivity (900-fold in binding and >10 000-fold in FLIPR assays) for OX2 over OX1 receptors. Moreover, the selective OX2 and dual OX1/OX2 receptor antagonists were able to competitively displace [3H]EMPA binding from hOX2-HEK293 membranes with the following rank order of potency: EMPA > Cp-1 > almorexant > Cp-4 > Cp-2 > Cp-3 > Cp-5. The selective OX1 receptor antagonists, SB 334867 and SB 674042 only poorly displaced [3H]EMPA binding from OX2-HEK293 membranes (Ki values >10 000 and 334 nmol·L−1 respectively).

    In functional studies, EMPA inhibited orexin-A- and orexin-B-induced [Ca2+]i responses in a CHO(dHFr-)-hOX2 stable cell line with a Kb value of 0.8 or 0.6 nmol·L−1 respectively. Analysis of the antagonism by EMPA, using orexin-A- or orexin-B-evoked accumulation of [3H]IP assay in CHO(dHFr-)-hOX2 cells, revealed that it behaved as a competitive antagonist with a Schild slope close to unity. Therefore, EMPA antagonized orexin-A and orexin-B with similar potency in hOX2 receptor expressing cells and there was a good correlation between EMPA's affinity constant (Ki) and apparent antagonist potency (Kba). Moreover, EMPA was assessed over a battery of 80 different binding sites that included numerous other GPCRs, transporters and ion channels. The results obtained indicate that EMPA displays a high degree of selectivity for OX2 receptors

    The property of the radioligand [3H]EMPA to selectively bind in vitro to rat brain sections was investigated. This study is the first to describe the regional binding properties of a radiolabelled selective antagonist for OX2 receptors in rat brain. High density of specific binding was observed in the CA3 region of the hippocampus, cortical layer 6, tuberomammillary nucleus, induseum griseum and nucleus accumbens. Binding was also observed in various thalamic and hypothalamic nuclei known to express OX2 receptors, such as the dorsal raphe and the pontine gray. Binding of [3H]EMPA to brain sections was completely abolished by the addition of the non-radiolabelled dual antagonist, almorexant, or selective OX2 receptor antagonists (CP-5 and EMPA). The ex vivo[3H]EMPA binding studies allowed determination of OX2 receptor occupancy by almorexant in rat brain regions. When given i.p., almorexant dose-dependently increased OX2 receptor occupancy, as measured by displacement of [3H]EMPA, with a dose of 30 mg·kg−1 producing 60–70% occupancy in rat brain. These results confirm the selectivity of [3H]EMPA for OX2 receptors. Furthermore, the distribution of binding sites for [3H]EMPA in coronal sections of rat brain were in good correlation with that of OX2 receptor transcripts and protein studied by hybridization histochemistry (Trivedi et al., 1998; Marcus et al., 2001) and IHC (Cluderay et al., 2002). The high density of [3H]EMPA binding in cortical layer 6 is in good agreement with a previous report showing the exclusive postsynaptic excitatory action of orexin on sublayer 6b cortical neurons via its interaction with OX2 receptors (Bayer et al., 2004). It is interesting to note that the highest binding density in the nucleus accumbens was observed in the shell yet only moderate binding in the core. In this context, the lateral hypothalamic orexin neurons project to reward-associated brain regions that include the nucleus accumbens and the ventral tegmental area suggesting the involvement of orexins in drug-seeking and other motivational behaviours (Harris et al., 2005; Scammell and Saper, 2005). Indeed, the infusion of orexin-A into the nucleus accumbens shell in rat stimulated both feeding and LMA (Thorpe and Kotz, 2005) while the preadministration of SB-334867-A (an OX1 receptor selective antagonist) significantly attenuated this orexin-A-induced feeding activity but had no effect on orexin-A-augmented LMA. As OX2 receptors are expressed in nucleus accumbens to a greater extent than are OX1 receptors (Lu et al., 2000; Cluderay et al., 2002), it is plausible that the effects on LMA are mediated via OX2 receptors. Furthermore, it has recently been shown that orexin-A activation of the nucleus accumbens shell acted as a mediator in the expression of precipitated morphine withdrawal (Sharf et al., 2008).

    The single-dose pharmacokinetic profiles of EMPA were assessed in mice and rats after intravenous and oral administration. EMPA displayed a high systemic plasma clearance, medium volume of distribution at steady state and low oral bioavailability in both mouse and rat. The mean brain/plasma concentration ratio of EMPA (at dose of 18 mg·kg−1, p.o.) was 0.2 in mouse. Because of lower oral bioavailability of EMPA in rat (F = 1.1%) in comparison to mouse (F = 21.7%), the brain level of EMPA in rat (at dose of 20 mg·kg−1, p.o.) was <10 ng·mL−1. Moreover, the plasma and brain exposure of EMPA was evaluated at the end of rodent behavioural procedures: the mean brain/plasma concentration ratios of EMPA determined at 45 and 65 min after i.p. administration were 0.7 and 0.1 in mouse and rat respectively.

    Orexin levels fluctuated diurnally in freely moving rats with levels slowly increasing during active phase and decreased during the rest (Taheri et al., 2000; Yoshida et al., 2001; Desarnaud et al., 2004). Moreover, i.c.v. injection of orexin-A during the resting phase increased arousal, grooming and LMA (Ida et al., 2000; Nakamura et al., 2000; Piper et al., 2000). In the present study, a selective OX2 receptor agonist, [Ala11,D-Leu15]orexin-B, which had shown a 400-fold selectivity for the OX2 (EC50= 0.13 nmol·L−1) over OX1 receptors (EC50= 52 nmol·L−1) (Asahi et al., 2003) was used to better address EMPA's selectivity for OX2 receptors in vivo. During the resting phase, i.c.v. injection of [Ala11,D-Leu15]orexin-B in mice significantly induced hyperlocomotor activity relative to vehicle. Although EMPA by itself had no effect on spontaneous LMA in freely moving mice, it significantly reversed [Ala11,D-Leu15]orexin-B-induced hyperlocomotion in a dose-dependent manner during the resting phase. Furthermore, EMPA, given in rats during the active phase, reduced significantly the LMA in a dose-dependent manner. Of note was the significant inhibition in spontaneous activity, compared with the vehicle treated animal, when LMA was summated over a 10 min period after 30 mg·kg−1 i.p. administration of EMPA, this despite a low brain to plasma ratio of EMPA and limited bioavailability in rat. As the OX2 receptor is expressed at a low level in rat peripheral tissues (Voisin et al., 2003; Heinonen et al., 2008) and anatomical, neurochemical and behavioural studies in rodents all support a central role of endogenous orexin in regulating motor activity (Peyron et al., 1998; Piper et al., 2000; Baldo et al., 2003; Krout et al., 2003), the observed cerebral level of 8 ng·mL−1 that is equivalent to a 18 nmol·L−1 concentration or more precisely 1.8 nmol·L−1 unbound EMPA in rat brain was nevertheless sufficient to exert this effect centrally, due to EMPA's high potency (Kb= 0.6 nmol·L−1) and fraction unbound (Fu = 10%). However, EMPA did not induce any deficits in the rat rotarod performance procedure for motor coordination and balance. The robust activity of EMPA in reversal of hyperlocomotion induced by a selective OX2 receptor agonist further substantiates the in vivo selectivity of EMPA for the OX2 receptor that is in good agreement with the high degree of selectivity observed in vitro. In conclusion, EMPA is a high-affinity, reversible, selective and in vivo active OX2 receptor antagonist. Thus, EMPA could prove useful when investigating the role played by OX2 receptors in pathophysiological processes of CNS disorders such as insomnia, cluster headache, drug abuse and maladaptation to stress.


    We are grateful to Patricia Glaentzlin, Valérie Goetschy, Claudia Kratzeisen, Anne Marcuz, Marie-Thérèse Miss, Céline Sutter and Marie-Thérèse Zenner for their excellent technical assistance.

      Conflicts of interest

      All authors are employees of F. Hoffmann-La Roche Ltd.