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Neuropharmacology

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Drug-receptor kinetics and sigma-1 receptor affinity differentiate clinicallyevaluated histamine H3 receptor antagonistsDarren M. Riddya, Anna E. Cooka, David M. Shackleforda, Tracie L. Piercea, Elisabeth Mocaerb,Clotilde Mannoury la Courb, Aurore Sorsb, William N. Charmana, Roger J. Summersa,Patrick M. Sextona, Arthur Christopoulosa, Christopher J. Langmeada,∗

a Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Victoria, Australiab Institut de Recherches Internationales Servier, 50 Rue Carnot, 92284, Suresnes, France

H I G H L I G H T S

• H3R receptor antagonists display differential kinetic properties.

• S 38093-2 H3R affinity at odds with in vivo activity in models of cognition.

• S 38093-2 preferentially occupies the mouse sigma-1 receptor in vivo.

• Sigma-1 affinity may play a role in H3R antagonists' clinical efficacy.

A R T I C L E I N F O

Keywords:Histamine H3 receptorSigma-1 receptorKineticsReceptor occupancyS 38093-2

A B S T R A C T

The histamine H3 receptor is a G protein-coupled receptor (GPCR) drug target that is highly expressed in theCNS, where it acts as both an auto- and hetero-receptor to regulate neurotransmission. As such, it has beenconsidered as a relevant target in disorders as varied as Alzheimer's disease, schizophrenia, neuropathic pain andattention deficit hyperactivity disorder. A range of competitive antagonists/inverse agonists have progressedinto clinical development, with pitolisant approved for the treatment of narcolepsy. Given the breadth ofcompounds developed and potential therapeutic indications, we assessed the comparative pharmacology of sixinvestigational histamine H3 agents, including pitolisant, using native tissue and recombinant cells. Whilst all ofthe compounds tested displayed robust histamine H3 receptor inverse agonism and did not differentiate betweenthe main H3 receptor splice variants, they displayed a wide range of affinities and kinetic properties, and in-cluded rapidly dissociating (pitolisant, S 38093-2, ABT-239) and slowly dissociating (GSK189254, JNJ-5207852,PF-3654746) agents. S 38093-2 had the lowest histamine H3 receptor affinity (pKB values 5.7–6.2), seemingly atodds with previously reported, potent in vivo activity in models of cognition. We show here that at pro-cognitiveand anti-hyperalgesic/anti-allodynic doses, S 38093-2 preferentially occupies the mouse sigma-1 receptor invivo, only engaging the histamine H3 receptor at doses associated with wakefulness promotion and neuro-transmitter (histamine, ACh) release. Furthermore, pitolisant, ABT-239 and PF-3654746 also displayed appre-ciable sigma-1 receptor affinity, suggesting that this property differentiates clinically evaluated histamine H3

receptor antagonists and may play a role in their efficacy.

https://doi.org/10.1016/j.neuropharm.2018.10.028Received 21 June 2018; Received in revised form 18 September 2018; Accepted 19 October 2018

Abbreviations: N-α-methylhistamine dihydrochloride, NαMH; R-α-methylhistamine dihydrochloride, RαMH; Haloperidol, 2-(1H-imidazol-5-yl)ethyl imidothio-carbamate, imetit; 4-(2-{2-[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile, ABT-239; 1-{3-[3-(4-chlorophenyl)propoxy]propyl}piperidine, pito-lisant; trans-N-Ethyl-3-fluoro-3-[3-fluoro-4-(1-pyrrolidinylmethyl)phenyl]-cyclobutanecarboxamide 4-methylbenzenesulfonate, PF-3654746; 1-{3-[4-(Piperidin-1-ylmethyl)phenoxy]propyl}piperidine, JNJ-5207852; 6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]eN-methyl-3-pyridinecarboxamide,GSK189254; 4-(3-(hexahydrocyclopental[c]pyrrol-2(1H)-yl)propoxy0benzamide, S 38093-2; room temperature, RT

∗ Corresponding author. Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC, 3052,Australia.

E-mail address: chris.langmead@monash.edu (C.J. Langmead).

Neuropharmacology 144 (2019) 244–255

Available online 22 October 20180028-3908/ © 2018 Published by Elsevier Ltd.

T

1. Introduction

The histamine H3 receptor is one of four histamine receptor sub-types categorised as Family A class of G protein-coupled receptors(GPCRs). Histamine receptors make excellent drug targets, with H1 andH2 receptor antagonists developed for the treatment of allergies andgastroesophageal reflux disorder, respectively. The histamine H3 re-ceptor is primarily expressed in the CNS and is recognised as a pre-synaptic auto-receptor (Arrang et al., 1983) and a hetero-receptor,regulating neurotransmitters such as acetylcholine (Arrang et al.,1995), dopamine (Ellenbroek, 2013), noradrenaline (Schlicker et al.,1990) and serotonin (Threlfell et al., 2004). Furthermore, recent studieshave shown that the H3 receptor can modulate both glutamate andGABA transmission (Schlicker and Kathmann, 2017; Nieto-Alamillaet al., 2016). The receptor is widely expressed in human brain with highlevels localized to areas associated with cognition, such as the striatum,cerebral cortex, and hippocampus (Pillot et al., 2002). Due to its loca-tion and pleiotropic ability to regulate neurotransmission, the H3 re-ceptor has been suggested as a potential drug target for the treatment ofAlzheimer's disease, schizophrenia, sleep-wake disorders, neuropathicand osteoarthritic pain, epilepsy, obesity and attention-deficit-hyper-activity disorder. With such a broad range of potential therapeutic in-dications and the prior successes in developing ligands targeting otherhistamine receptor subtypes, it was natural that researchers sought todevelop H3 receptor selective antagonists.

Compared to the other histamine receptor subtypes, the H3 receptorexhibits rather complex pharmacology, being subject to extensivesplicing, leading to two major (H3-445 and H3-365) and possibly morefunctional human receptor isoforms; for a comprehensive review pleasesee Leurs et al. (2005) and Bongers et al. (2007). The receptor is alsoreportedly to be constitutively active to various degrees, renderingmechanistic understanding of the pharmacology of “antagonists” aspartial agonists, neutral antagonists and inverse agonists essential, asdifferential activity can yield different functional or “protean” out-comes (Gbahou et al., 2003; Krueger et al., 2005).

Despite these challenges, multiple compounds have progressed intoclinical development (exemplified in Fig. 1) for a range of target in-dications, including Alzheimer's disease, neuropathic pain, narcolepsy,and excessive daytime sleepiness. Pitolisant (also known as tiprolisant,ciproxidine or BF 2649) was the first H3 receptor antagonist to progressinto clinical development, ultimately being approved for the treatmentof narcolepsy (with further Phase III trial results pending; Abad andGuilleminault, 2017; Wozniak and Quinnell, 2015). Other agents, in-cluding ABT-239, GSK189254, S 38093-2, JNJ-5207852 and PF-3654746, progressed into either Phase I or Phase II trials but werediscontinued due to a lack of efficacy, insufficient safety or a strategicwithdrawal from neuroscience R&D (Kubo et al., 2015; Lazewska and

Kiec-Kononowicz, 2018).Although each and every one of the agents listed above has been

independently described in the literature by the originating labora-tories, the extent to which their comparative receptor pharmacologymight underlie their varied efficacy profile across different pre-clinicalmodels and clinical populations remains relatively unexplored.Accordingly, we examined the pharmacology of the aforementionedinvestigational and approved histamine H3 receptor antagonists tocompare their affinities, inverse agonist efficacies and any differentialisoform activity at H3-445 and H3-365 utilising cAMP accumulation,GTPγ35S binding and intracellular Ca2+ mobilization in recombinantcells and/or rat striatal tissue. We show that all H3 receptor antagonistsexhibit inverse agonist activity in membranes prepared from re-combinant H3 receptor expressing CHO cells, and use functional kineticassays (Riddy et al., 2015; Christopoulos et al., 1999; Mathiesen et al.,2006) to demonstrate that these compounds fall broadly into two dis-tinct kinetic classes: “slow-off rate” antagonists (GSK189254, JNJ-5207852 and PF-3654746) and “fast-off rate” antagonists (ABT-239,pitolisant and S 38093-2), properties that do not consistently correlatewith their equilibrium affinity.

Histamine H3 antagonists including thioperamide and iodophen-propit display sub-micromolar affinity for other receptors, including thesigma-1 receptor (Leurs et al., 1995). Although not a G protein-coupledreceptor, sigma-1 receptors are known to regulate ion channel function,including NMDA/GluN receptors and K+/Ca2+ channels (Cobos et al.,2008). Antagonists of this receptor are effective in models of neuro-pathic pain (Abadias et al., 2013; Zamanillo et al., 2013) and off targetprofiling of S 38093-2 revealed modest activity at the peripheral sigma-1 receptor. Due to this activity we hypothesized that part of the ob-served in vivo activity of H3 antagonists, including S 38093-2, may bedue to a combination of both H3 and sigma-1 activities.

Using in vitro [3H]-(+)-pentazocine binding and ex vivo receptoroccupancy studies we demonstrate that S 38093-2 preferentially occu-pies sigma-1, but not histamine H3 receptors, at cognition-enhancingdoses. Indeed, mouse brain histamine H3 versus sigma-1 affinity ratiosalso differentiated the other agents tested; GSK189254 and JNJ-5207852 display high histamine H3 receptor selectivity, whereas PF-3654746, ABT-239 and pitolisant have similar histamine H3 and sigma-1 receptor affinity, suggesting that the latter property could contributeto their in vivo efficacy.

2. Materials and methods

2.1. Cell line generation and culture

Stably expressed, tetracycline-inducible, Flp-IneCHO-TREx H3R celllines were generated as previously described by Riddy et al. (2017).Clones were functionally screened for high expression as determined bythe efficacy of imetit 1 μg per mL tetracycline-treated cells in the in-hibition of cAMP accumulation assay. Cell lines were maintained inDMEM: Ham's F-12 1:1 supplemented with 16 mM HEPES, 5 μg per mLblasticidin, 300 μg per mL hygromycin B and 10% tetracycline free FBSat 37 °C/5% CO2 and induced overnight with 1 μg per mL tetracycline.

2.2. Recombinant cell and striatal rat brain membrane preparation

Cells were grown to 80–90% confluence in 500 cm2 cell-culturetrays at 37 °C/5% CO2. All subsequent steps were conducted at 4 °C toavoid receptor degradation. The cell-culture medium was removed andice-cold buffer (10 mL per tray; 10 mM HEPES, 0.9% w/v NaCl, 0.2%w/v EDTA, pH 7.4) was added to the cells. The cells were scraped fromthe trays into a 50 mL Corning tube and centrifuged at 250×g for 5 min.The supernatant fraction was aspirated and 10 mL per 500 cm2 tray ofwash buffer (10 mM HEPES, 10 mM EDTA, pH 7.4) was added to thepellet. The pellet or striatal rat brain were prepared by homogenizationusing an electrical homogenizer ‘Werker, ultra-turrax’ (position 6,Fig. 1. Structures of histamine H3 receptor antagonists tested in these studies.

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4 × 5 s bursts) and centrifuged at 48,000×g at 4 °C (Beckman Avanti J-251 Ultracentrifuge) for 30 min. The supernatant was discarded and thepellet re-homogenised in wash buffer and centrifuged as describedabove. The final pellet was suspended in ice-cold assay buffer (10 mMHEPES, 0.1 mM EDTA, pH 7.4) at a concentration of 1–2 mg/mL.Protein concentration was determined by a bicinchoninic acid assay,using BSA as a standard and aliquots maintained at −80 °C until re-quired.

2.3. GTPγ35S binding

Ten-point concentration-response curves of the compounds wereprepared at 100x concentration in 100% DMSO, and 2.5 μL added to a96-well assay plate. For antagonist curves a concentration of R-α-me-thyl histamine (RαMH), corresponding to approximately an EC60 - EC 80

concentration, prepared in DMSO, was added in a volume of 2.5 μL perwell. To each well, 200 μL of assay buffer, with 0.1% BSA and 30 μg permL saponin (added fresh on the day of experimentation), containing50 μg per mL membranes, and 3.7 μM guanosine 5‘-diphosphate sodiumsalt (GDP) was added and incubated at room temperature with gentleagitation for 30 min, allowing equilibrium to be reached. Followingthis, 50 μL of [35S]-guanosine 5'-(γ-thio)triphosphate (GTPγ35S) at afinal concentration of 300 pM was added to each well and incubated atroom temperature (RT) with gentle agitation for 40 min. After the in-dicated incubation period, bound and free GTPγ35S were separated byrapid vacuum filtration using a FilterMate™ Cell Harvester (PerkinElmer, Melbourne, Australia) onto 96-well GF/C filter plates and ra-pidly washed three times with ice cold wash buffer (50 mM Tris-HCL,10 mM MgCl2, 100 mM NaCl, pH 7.6). After drying (> 4 h), 40 μL ofMicroscint™ 20 (Perkin Elmer, Melbourne, Australia) was added to eachwell and radioactivity quantified using single photon counting on aMicroBeta2 microplate scintillation counter (Perkin Elmer, Melbourne,Australia).

2.4. Cyclic AMP accumulation

The accumulation of cAMP was measured using an AlphaScreen™SureFire™ cAMP Assay Kit (Perkin-Elmer). Briefly, cells were seededinto transparent 96-well plates at 40,000 cells per well in the presenceof 1 μg per mL tetracycline and were incubated overnight at 37 °C with5% CO2. Cells were washed twice with PBS and incubated in stimula-tion buffer (phenol red-free DMEM (GIBCO), 1% BSA and 1 mM 3-iso-butyl-1-methylxanthine), at 37 °C with 5% CO2 for 1 h. For determi-nation of agonist stimulated concentration-response curves, cells werestimulated with compounds of interest for 10 min at 37 °C with 5% CO2

prior to addition of 3.16 μM forskolin and incubation at 37 °C with 5%CO2 for a further 20 min. Media were removed and reactions termi-nated by addition of 50 μL per well ice cold 100% ethanol. Cells wereincubated at RT with shaking for 5 min prior to removal of ethanol byevaporation. Cells were rehydrated using 100 μL per well lysis buffer(0.1% BSA, 5 mM HEPES, 0.3% Tween) with shaking for 5 min at RT.For each well, 5 μL per well lysate was transferred to an opaque 384-plate. AlphaScreen™ acceptor and biotin loaded donor bead mixes werediluted according to the manufacturers protocol in lysis buffer andadded to the lysates and allowed to incubate overnight in the dark atRT. Fluorescence was measured using a Fusion plate reader(PerkinElmer, MA).

2.5. [Ca2+]i-mobilization assays

Assays were performed in isotonic buffer (150 mM NaCl, 2.6 mMKCl, 2.2 mM CaCl2, 1.18 mM MgCl2, 10 mM D-glucose, 10 mM HEPES,4 mM probenecid and 0.5% w/v BSA, pH 7.4). Assay buffer was pre-pared as a [10x] stock (without probenecid or BSA), sterile filtered andstored at 4 °C until required. A [1x] stock (500 mL) of assay buffer wasprepared by adding 50 mL of 10x stock and 5 mL of 400 mM probenecid

(dissolved in 1 M NaOH) to 440 mL H20, and pH adjusted to 7.4, beforemaking up the final volume to 500 mL. BSA (0.5% w/v) was added and[1x] assay buffer stored at 4 °C for up to two weeks.

Cells were seeded onto clear 96-well plates in growth medium at adensity of 40,000 cells per well and treated with 1 μg per mL finaltetracycline. The next day, plates containing a confluent monolayer ofcells were twice washed with 100 μL assay buffer, before being loadedwith 100 μL assay buffer containing 0.9 μM Fluo-4-AM. After 1 h in-cubation at 37 °C, Fluo-4-AM containing buffer was removed and wellswere twice washed with 100 μL assay buffer, before being loaded with180 μL assay buffer. After 10–20 min, the addition of compounds(agonist-mode screen) was added on a Flexstation 1 or 3 microplatereader (Molecular Devices; Sunnyvale, California). Peak fluorescencewas detected using 485 nm excitation and 525 nm emission filters. Datawere normalised to the response to 100 μM ATP (positive control forlive cell number). Inverse agonists/antagonists were pre-incubated for20 min following Fluo-4-AM washout. This assay format was utilised toestimate ligand dissociation rates (Riddy et al., 2015) for all inverseagonists/antagonists (see Data Analysis).

2.6. Sigma receptor function (field-stimulated Guinea-pig vas deferens)

Sigma receptor antagonist activity was detected by inhibition ofPRE-084 (100 μM)-stimulated twitch contraction by S 38093-2 or BD1047 (control sigma receptor antagonist) as per Vaupel and Su (1987).Data are expressed as percentage of the effect mediated by PRE-084(100 μM; n = 8). Segments of guinea pig vas deferens were suspendedin 20 mL organ baths filled with an oxygenated (95% O2 and 5% CO2)and pre-warmed (37 °C) physiological salt solution of the followingcomposition: NaCl 118.0 mM, KCl 4.7 mM, MgSO4 1.2 mM, CaCl22.5 mM, KH2PO4 1.2 mM, NaHCO3 25.0 mM and glucose 11.0 mM (pH7.4). Yohimbine (1 μM), propranolol (1 μM), (−)sulpiride (1 μM), ci-metidine (1 μM), atropine (1 μM), naloxone (1 μM) and methysergide(1 μM) were also present throughout the experiments to block the α2-adrenergic, β-adrenergic, dopamine D2, histamine H2, muscarinic,opioid, and 5-HT1/5-HT2 receptors, respectively. The tissues wereconnected to force transducers for isometric tension recordings. Theywere stretched to a resting tension of 0.5 g then allowed to equilibratefor 60 min during which time they were washed repeatedly and thetension readjusted. Thereafter, they were stimulated electrically with1 s trains of square wave pulses (maximal intensity, 1 ms duration,5 Hz) delivered at 10 s intervals by a constant current stimulator. Theexperiments were carried out using semi-automated isolated organsystems possessing eight organ baths, with multichannel data acquisi-tion.

2.7. In vitro [3H]-(+)-pentazocine/[3H]eN-α-methyl histamine (NαMH)mouse brain binding

All radioligand experiments were conducted in 10 mL tubes, inassay binding buffer (50 mM Tris-HCL, 5 mM EDTA, pH 7.7) at 25 °C.Frozen whole Swiss mouse brain was rapidly defrosted and homo-genised in 55 mL w/v assay binding buffer; homogenates (2 mg/well)were incubated with assay buffer or varying concentration of test li-gand. Non-specific binding (NSB) was determined in the presence of10 μM haloperidol ([3H]-(+)-pentazocine binding) or 10 μM ABT-239([3H]-NαMH binding). After 1 h incubation, bound and free radioligandwere separated by rapid vacuum filtration using a FilterMate™ CellHarvester (Perkin Elmer, Melbourne, Australia) onto 96-well GF/B filtermats presoaked with 0.3% PEI and rapidly washed three times with icecold wash buffer (50 mM Tris-HCl, 5 mM EDTA, pH 7.7). After drying(> 1 h), filter mats were separated and placed into scintillation vials.To these 2 mL of Ultima Gold (Perkin Elmer, Melbourne, Australia) wasadded and radioactivity quantified using a liquid scintillation spectro-metry, LS 6500 scintillation counter (Beckman Coulter, High Wycombe,UK). Aliquots of radioligand were also quantified accurately to

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determine how much radioactivity was added to each well. In all ex-periments, total binding never exceeded more than 10% of that added,limiting complications associated with depletion of the free radioligandconcentration.

2.8. Plasma and brain exposure of S 38093-2 and GSK189254 in mice

S 38093-2 was dissolved in saline at a concentration of 1 mg/mL(target dose of 10 mg/kg) producing a clear solution. S 38093-2 for-mulations to achieve lower doses of 0.3, 1 and 3 mg/kg were preparedby means of serial dilution in saline; tubes were inverted prior todrawing each dosing volume to mix the bulk formulations. GSK189254was wet milled in 1% (w/v) methylcellulose in an agate mortar andpestle and sonicated to produce a uniform, almost clear suspension. Thebulk formulation was mixed by inverting the tube prior to drawing eachdosing volume and animals dosed within 1 h of preparation.

All animal studies were conducted using established procedures inaccordance with the Australian Code of Practice for the Care and Use ofAnimals for Scientific Purposes, and the study protocols were reviewedand approved by the Monash Institute of Pharmaceutical SciencesAnimal Ethics Committee. Male Swiss outbred mice, aged between 6-8-weeks, were used for experiments. Mice were grouped-housed in a 12-hlight, 12-h dark cycle with standard rodent chow and water ad libitum.

Brain exposure and ex vivo histamine H3 and sigma-1 receptorbinding (vide infra) of S 38093-2 and GSK189254 following PO ad-ministration were studied in non-fasted mice. Compounds were ad-ministered by injection or orally by gavage (10 mL per kg dose volume),and a single blood sample was collected from each mouse (via cardiacpuncture while anaesthetised using gaseous isoflurane) 15 min post-dose (n = 6 mice at each dose level). At the time of blood sampling, andwhile still under anaesthesia, each mouse was killed by cervical dis-location, and the whole brain was rapidly removed. The whole brainswere then blotted to remove excess blood and divided into two halves.The left hemispheres were snap frozen and used for ex vivo receptoroccupancy studies (Section 2.9). The right hemispheres were weighed,snap frozen in dry ice and subsequently stored frozen (−20 °C) until LC-MS analysis with a Micromass Quattro Premier coupled to a WatersAcquity UPLC using positive electrospray ionisation multiple-reactionmonitoring mode for detection. Analyte quantitation was performedrelative to calibrations standards prepared in blank brain homogenate.

2.9. CSF exposure of S 38093-2 in rats

S 38093-2 was dissolved in saline at concentrations appropriate toyield final doses of 0.3 or 3 mg/kg in a final dose volume of 3 mg/mL.CSF levels of S 38093-2 were monitored after oral drug administrationto male Wistar rats (250–300 g; n = 3/dose) by collection from a ca-theter implanted in the cisterna magna. CSF was transferred into anEppendorf tube and stored at −60 °C or below until bioanalysis. CSFsamples were thawed at room temperature and agitated vertically for1 min then centrifuged 5 min at 1000 rpm 20 μL of samples were addedinto the wells of an Ostro 96-well plate. Internal standard solution wasadded (Y 1789, 10 μL at 1 ng/mL + 0.3% of HCOOH). Samples wereshaken 1 min at 1500 rpm before injection. S 38093-2 in CSF wasmeasured by liquid chromatography (column C18 CSH 50 × 2.1 mm,1.7 μm) using tandem mass spectrometry detection (API 4000) in ESI+,using Y 1789 as internal standard. The working range for CSF was0.05–50 ng/mL using 20 μL of CSF.

2.10. Ex vivo mouse brain receptor occupancy

Individual brain hemispheres isolated from animals post oral ad-ministration of either S 38093-2 or GSK189254 were homogenised inice-cold assay buffer as for radioligand binding above to give a finalassay concentration of 5.4 mg/mL. Non-specific binding (NSB) wasdetermined in the presence of 10 μM haloperidol ([3H]-

(+)-pentazocine binding) or 10 μM ABT-239 ([3H]-NαMH binding).After 1 h incubation, bound and free radioligand were separated byrapid vacuum filtration using a FilterMate™ Cell Harvester (PerkinElmer, Melbourne, Australia) onto 96-well GF/B filter mats presoakedwith 0.3% PEI and rapidly washed three times with ice cold wash buffer(50 mM Tris-HCl, 5 mM EDTA, pH 7.7). After drying (> 1 h), filter matswere separated and placed into scintillation vials. To these 2 mL ofUltima Gold (Perkin Elmer, Melbourne, Australia) was added andradioactivity quantified using a liquid scintillation spectrometry, LS6500 scintillation counter (Beckman Coulter, High Wycombe, UK).Aliquots of radioligand were also quantified accurately to determinehow much radioactivity was added to each well. In all experiments,total binding never exceeded more than 10% of that added, limitingcomplications associated with depletion of the free radioligand con-centration. Total and non-specific binding of both [3H]-(+)-pentazo-cine and [3H]-NαMH binding were determined in sextuplicate for eachbrain sample; percentage of histamine H3 or sigma-1 receptor occu-pancy by S 38093-2 or GSK189254 was calculated based on differencein levels of specific binding between samples from vehicle and drugtreated groups.

2.11. Plasma protein and mouse brain tissue binding

Mouse plasma from male Swiss outbred mice was procured fromAnimal Resources Centre (Perth) and was stored frozen at −80 °C. Onthe day of the experiment, frozen plasma was thawed in a water bathmaintained at 37 °C. Brain tissue from male Swiss outbred mice washarvested in house and was stored frozen at −80 °C. On the day of theexperiment, brain tissue was homogenised (gentleMACSTM Dissociator,Miltenyi Biotec GmbH, Germany) in 8 parts 0.1 M phosphate buffer (pH7.4) to 1-part brain tissue (assuming 1 g brain tissue is equal to 1 mL) toachieve a fine suspension with a tissue concentration of 0.11 g tissue/mL (9-fold dilution). Aliquots of mouse plasma and brain tissuehomogenate were spiked with a DMSO/acetonitrile/water solution ofeach test compound; 2000 ng/mL for plasma and 1000 ng/g tissue forbrain (maximum final DMSO and acetonitrile concentrations were 0.2%(v/v) and 0.4% (v/v), respectively). Aliquots (n = 3) of spiked matrixwere immediately frozen (−20 °C), and the remaining volume was in-cubated at 37 °C (60 min for plasma and 10 min for brain tissuehomogenate) to equilibrate; aliquots (n = 5 for plasma and n = 4 forbrain tissue homogenate) of spiked matrix were transferred into tubesand subjected to ultracentrifugation (Beckman Rotor type 42.2 Ti;223,000×g and 37 °C) for 4.2 h. Additional aliquots (n = 3) of spikedbrain homogenate were maintained at 37 °C over the course of ultra-centrifugation to obtain a measure of the total test compound con-centration (Ctotal). Following ultracentrifugation, an aliquot of super-natant was taken from each of n = 4 ultracentrifuge tubes to obtainmeasures of the unbound concentration (Cunbound) in each matrix. Forplasma, the contents of the remaining ultracentrifuge tube were mixedto redistribute the protein prior to taking aliquots (n = 3) to obtain ameasure of the total plasma compound concentration (Ctotal). All sam-ples were stored frozen at −20 °C until analysis. Concentrations of eachcompound in non-centrifuged matrix and protein-free supernatantsamples were determined by UPLC-MS (Waters/Micromass Xevo triplequadrupole mass spectrometer) relative to calibration standards pre-pared in corresponding blank matrices. The lower limits of quantitationfor the assay of the compounds ranged from 0.5 to 5 ng/mL for plasmaand 1–5 ng/mL for brain tissue homogenate. Fraction unbound inplasma was determined as the ratio of (Cunbound) to (Ctotal); brain tissuebinding analysis was as per Kalvass and Maurer (2002).

2.12. Compounds and reagents

All compounds were supplied from Sigma Aldrich, Castle Hill, Australia(R-α-methylhistamine dihydrochloride, Haloperidol); or Servier, Paris,France (2-(1H-imidazole-5-yl)ethyl imidothiocarbamate; imetit, 4-(2-{2-

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[(2R)-2-Methylpyrrolidin-1-yl]ethyl}-benzofuran-5-yl)benzonitrile; ABT-239, 1-{3-[3-(4-chlorophenyl)propoxy]propyl}piperidine; pitolisant, trans-N-Ethyl-3-fluoro-3-[3-fluoro-4-(1-pyrrolidinylmethyl)phenyl]-cyclobutane-carboxamide 4-methylbenzenesulfonate; PF-3654746, 1-{3-[4-(Piperidin-1-ylmethyl)phenoxy]propyl}piperidine; JNJ-5207852, 6-[(3-Cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]eN-methyl-3-pyridinecarbox-amide; GSK189254, and 4-(3-(hexahydrocyclopental[c]pyrrol-2(1H)-yl)propoxy0benzamide; S 38093-2. Radioligands were supplied by PerkinElmer, Melbourne, Australia. Cell culture and molecular biology reagentswere supplied by Life Technologies, Melbourne, Australia.

2.13. Data analysis

All values reported in the text and tables are mean ± SEM for theindicated number of experiments. All experiments were analysed byeither linear or nonlinear regression using Prism 6.0 or 7.0 (GraphPadSoftware Inc., San Diego, CA).

Inhibition curves from binding or functional assays were fitted tosigmoidal (variable slope) curves using a four-parameter logisticequation:

= ++

Y Bottom Top BottomLogIC X Hillslope( – )

1 10ˆ (( 50 ) )

Where Y is the % bound of radioligand or % response. Top and Bottomdenote the maximal and minimal asymptotes, respectively.

IC50 values obtained from antagonist inhibition curves were con-verted to KB values using the method of Leff and Dougall (1993):

=+

K ICA EC n n

[ ](2 ([ ]/[ ]) )1/ 1B

50

50

IC50 and EC50 are the concentrations at the midpoint of the inhibitorand agonist concentration-response curves, respectively, [A] is the fixedconcentration of agonist used and n is the slope factor for the curves,fitted globally.

Agonist concentration-response curves were fitted to sigmoidal(variable slope) using either a three- or four-parameter logistic equa-tion:

= ++

Y Bottom Top BottomLogEC X n

( – )1 10ˆ (( 50 )· )

Where Y is the response measured, Top and Bottom denote the maximaland minimal asymptotes, respectively, and ‘n’ represents the Hill slope(constrained to unity where appropriate).

For Ca2+ mobilization assays run under hemi-equilibrium condi-tions, a combined operational and hemi-equilibrium model for com-petitive antagonism under non-equilibrium conditions (Kenakin, 2009;Riddy et al., 2015) was fitted to the data to generate estimates of liganddissociation rate as follows:

=+ +B K

B K A K[ ]/

([ ]/ [ ]/ 1)B

B A

=+

B KB K

[ ]/([ ]/ 1)

B

B

= + ++

B K A KA K

([ ]/ [ ]/ 1)([ ]/ 1)

B A

A

= ++ + +

Y A K e e EA K e e

[ ]/ (1 ( ·(1 ) · ))· ·[ ]/ ((1 ( ·(1 ) · ))· 1) 1

Ak t k t

m

Ak t k t

· · · ·

· · · ·

off off

off off

[A] and [B] represent the concentrations of agonist (RαMH) andantagonist, respectively, KA and KB represent the respective equilibriumdissociation constants, koff is the dissociation rate constant for the an-tagonist (min−1), t is the assay incubation time (min), τ is the opera-tional efficacy of RαMH (comprising cell- and agonist-dependentproperties) and Em is the maximal system response. All parameters are

shared across all datasets except t, which was fixed to the assay in-cubation time.

Where the test antagonist caused a significant depression of themaximal RαMH response, an F-test was performed to determine whe-ther the estimated value of koff was significantly different to zero (i.e.pseudo-irreversible antagonism). If so, the value of koff was quotedas < 0.01 min−1 in the Results (which is the limit of the dynamic rangeof an assay of this duration to detect off-rates; Kenakin, 2009). Con-versely, if the test antagonist did not depress the maximal the RαMHresponse such that the above model could converge, the dataset wasanalysed according to a modified form of the Gaddum/Schild equationsfor competitive antagonism:

= +

++

Y Bottom Top Bottom( )

1 A

n

10[ ]

LogEC BpA

s H50 1 [ ]

10 2

where Top represents the maximal asymptote of the curves, Bottomrepresents the minimum asymptote of the curves, LogEC50 representsthe logarithm of the agonist EC50 in the absence of antagonist, [A] re-presents the concentration of RαMH, [B] represents the concentrationof the antagonist, nH represents the Hill slope of the agonist curve, srepresents the Schild slope for the antagonist (constrained to unity),and pA2 represents the negative logarithm of the concentration of an-tagonist that shifts the agonist EC50 by a factor of 2 (which is reportedas a pKB as the Schild slope is fixed to one). For these datasets the valueof koff was quoted as > 4 min−1 in the Results to reflect the limit of thedynamic range of the Ca2+ mobilization assay to detect dissociationrates (Kenakin, 2009).

Estimated fractional histamine H3 or sigma-1 receptor occupancyfor S 38093-2 and GSK189254 (to compare with measured occupancyvalues in mouse) were calculated using total brain concentrations fromthe mouse PK (corrected for brain tissue binding) or rat CSF levels, andaffinity values from [3H]-NαMH and [3H]-(+)-pentazocine mousebrain homogenate binding, respectively:

= ×+

estimated receptor occupancy AA K

% 100 [ ][ ]

ub

ub B

where [A]ub is the free concentration in the brain and KB is the equi-librium dissociation constant of the compound for the target receptor.ROcc50 values (the dose of drug required to occupy 50% of the targetreceptor population) for S 38093-2 were calculated by fitting either theestimated or measured % receptor occupancy values against log [dose]using a three-parameter logistic function with maximum and minimumasymptotes constrained to 100 and 0, respectively.

Statistical significance was determined by Student's t-test with*P < 0.05, ***P < 0.001 and ****P < 0.0001 deemed significant.

3. Results

3.1. H3R antagonists exhibit a range of histamine H3 receptor affinities

The cohort of compounds shown in Fig. 1 displayed a wide range ofaffinities as measured by inhibition of R-α-methyl-histamine (RαMH)-stimulated GTPγ35S binding in CHO cells stably expressing the fulllength H3-445 receptor, with pKB values ranging from 9.9 ± 0.3(GSK189254) to as low as 6.2 ± 0.1 (S 38093-2) (Table 1; Fig. 2A). Allthe antagonists tested inhibited the 10 nM RαMH response to below thebasal level of GTPγ35S binding, suggesting that the receptor is con-stitutively active in this system and that the antagonists are all actuallyinverse agonists. Moreover, full inhibition of the agonist-stimulatedresponse with Hill slopes approximating unity suggests a competitivemechanism of action, and supports previous Schild analyses data for H3

antagonists (Esbenshade et al., 2005; Barbier et al., 2004).Inhibition of constitutive activity was confirmed using a cAMP

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accumulation assay in the same cells; whereas RαMH inhibited for-skolin (3 μM)-stimulated cAMP production, all of the antagonists in-creased cAMP accumulation in a concentration-dependent fashion, in-dicative of inverse agonist activity (Fig. 2B). Inverse agonist IC50 valueswere more potent than, but correlated with, pKB values from theGTPγ35S binding assay. In order to determine whether the antagonistpharmacology was preserved across species and in a native system(with a range of receptor splice variants likely present), the antagonistswere evaluated against RαMH-stimulated GTPγ35S binding in ratstriatal membranes. All of the compounds fully inhibited agonist-sti-mulated GTPγ35S binding with similar, if slightly lower, affinity esti-mates compared to recombinant studies (Table 1; Fig. 2C). However,limited inverse activity was observed compared to data generated usingmembranes from recombinant cell lines (Fig. 2A), implying reducedconstitutive receptor activity in native tissue.

Table 1Potency or affinity estimates of histamine H3 receptor antagonists/inverseagonists as determined in RαMH-stimulated GTPγ35S binding or constitutivecAMP production in Flp-IneCHO-TREx H3-445 cells or RαMH-stimulatedGTPγ35S binding in rat striatal membranes. Data are expressed as mean ± SEMfrom a fit to grouped data from n = 3–4 independent experiments.

Compound CHO TREx hH3-445GTPγ35S pKB

CHO TREx hH3-445cAMP pIC50

Rat striatumGTPγ35S pKB

JNJ-5207852 9.68 ± 0.13 9.40 ± 0.20 8.17 ± 0.17PF-3654746 8.78 ± 0.29 10.35 ± 0.80 8.29 ± 0.14GSK189254 9.92 ± 0.32 11.74 ± 0.60 9.64 ± 0.39ABT-239 9.20 ± 0.14 8.94 ± 0.18 8.82 ± 0.18Pitolisant 8.12 ± 0.09 9.56 ± 0.25 7.70 ± 0.24S 38093-2 6.19 ± 0.14 6.34 ± 0.25 6.12 ± 0.81

Fig. 2. In vitro compound affinities of histamine H3Rantagonists. (A) Concentration-dependent effects of R-α-methyl-histamine (RαMH) or JNJ-5207852, PF-3654746,GSK189254, ABT-239, pitolisant or S 38093-2 in the presenceof 10 nM RαMH in GTPγ35S binding in membranes preparedfrom Flp-IneCHO-TREx-H3-445 cells. (B) Concentration-de-pendent effects of the same compounds on forskolin (3 μM)-stimulated cAMP production in Flp-IneCHO-TREx H3-445 cells. (C) Concentration-dependent effects of R-α-methyl-histamine (RαMH) or test antagonists in the presence of10 nM of RαMH in GTPγ35S binding in membranes preparedfrom rat striatal tissue. Grouped data are shown ± SEM(n = 3–4).

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3.2. Histamine H3 receptor antagonists have different receptor residencetimes

Taking advantage of the non-equilibrium nature of intracellularCa2+ mobilization assays and applying a combined operational andhemi-equilibrium model for competitive antagonism under such con-ditions, we estimated the dissociation rate (koff) and affinity (pKB) foreach of the six histamine H3 receptor antagonists at both the H3-445and H3-365 isoforms. Affinity estimates for the antagonists were in linewith values obtained from the H3-445 GTPγ35S binding, with a generaltrend for the antagonists to display lower affinity for the H3-365 iso-form (Table 2). Despite the rather limited range of the functional assayto define dissociation rate, the estimates suggest that the compoundsfall broadly into two groups: rapidly dissociating antagonists (pitoli-sant, S 38093-2 and ABT-239) and slowly dissociating antagonists

(GSK189254 and PF-3654746; Table 2, Fig. 3). Interestingly, JNJ-5207852 displays a mixed profile, with seemingly slow dissociationfrom the full-length H3 receptor, but more rapid kinetics at the trun-cated isoform (Table 2). Furthermore, dissociation rate did not correlatestrongly with equilibrium affinity; pitolisant and PF-3654746 had si-milar pKB values (9.1 vs. 9.5) but markedly different estimated dis-sociation rates (1.53 vs. < 0.01 min−1).

3.3. Sigma-1 receptor binding differentiates clinical histamine H3 receptorantagonists

One of the clear discrepancies amongst the investigational hista-mine H3 antagonists is the very potent in vivo activity of S 38093-2 inmodels of cognition (minimum effective doses in the range 0.1–0.3 mg/kg) versus its low, micromolar affinity, and the much higher doses

Table 2Estimated equilibrium affinity constants (pKB), dissociation rate constants (koff) and receptor half-lives of H3 receptor antagonists as determined using a [Ca2+]I-mobilization assay under hemi-equilibrium conditions in Flp-IneCHO-TREx H3-365 or H3-445 cells. Data were analysed globally using the models/equations asdescribed in the Methods. Data are expressed as mean ± SEM from a fit to grouped data from n = 3–4 independent experiments.

Compound CHO TREx hH3-445 CHO TREx hH3-365

t½ (min) koff (min−1) pKB t½ (min) koff (min−1) pKB

JNJ-5207852 > 70 < 0.01 10.43 ± 0.12 9.9 0.07 ± 0.04 10.19 ± 0.13PF-3654746 > 70 < 0.01 9.53 ± 0.15 > 70 < 0.01 9.03 ± 0.13GSK189254 > 70 < 0.01 10.11 ± 0.15 > 70 < 0.01 10.22 ± 0.14ABT-239 2.5 0.27 ± 0.14 8.70 ± 0.18 5.0 0.14 ± 0.05 8.12 ± 0.09Pitolisant 0.5 1.53 ± 0.94 9.10 ± 0.16 < 0.25 > 4 8.22 ± 0.09S 38093-2 0.7 0.96 ± 0.96 6.12 ± 0.18 < 0.25 > 4 5.66 ± 0.15

Fig. 3. Kinetic rates of histamine H3R antagonists. Concentration response curves for R-α-methyl-histamine (RαMH), in the presence of increasing concentrationsof JNJ-5207852, PF-3654746, GSK189254, ABT-239, pitolisant or S 38093-2 in the [Ca2+]I-mobilization assay in Flp-IneCHO-TREx-H3-h365 or Flp-IneCHO-TREx-H3-h445 cells. Flp-IneCHO-TREx H3R cells were incubated at 37 °C with antagonist for 30 min prior to the addition of agonist and assayed with fluorescence beingmeasured over a 75 s time period using 485 nm excitation and 538 nm emission wavelengths. Data were analysed globally using the models/equations as described inthe Methods to calculate pKB and koff values for the test antagonists and are summarized in Table 2. Grouped data are shown ± SEM (n = 3–4).

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required to induce markers of histamine H3 receptor antagonism (e.g.cortical histamine and acetylcholine release at 10 mg/kg, antagonism ofRαMH-induced dispogenia from 10 mg/kg). Given the data above, thepotent in vivo activity in cognition models seems to be unlikely to be asa result of prolonged receptor residence time; S 38093-2 displayed theshortest half-life of all the histamine H3 receptor antagonists tested(Table 2).

S 38093-2 was subjected to a panel of potential off target GPCRsknown to be expressed in the brain to help rationalize properties thatmight underlie the compounds’ in vivo activity. Cross screening revealedno apparent off-target activity at any of the GPCRs tested(Supplementary Table A1); however, S 38093-2 displayed modest an-tagonist activity (pA2 = 6.2 ± 0.2, n = 2) at the sigma receptor, asmeasured by a functional assay in the guinea-pig vas deferens(Supplementary Fig. A).

Sigma-1 receptors are known to play a role in learning, memory andpain (Cobos et al., 2008; Nguyen et al., 2015; Zamanillo et al., 2013).Consequently, we evaluated S 38093-2 and the other H3 antagonists ina sigma-1 receptor, [3H]-(+)-pentazocine binding assay in mouse brainhomogenates, using the sigma-1 antagonist, haloperidol, as a positivecontrol. In parallel, we confirmed their affinity at the H3 receptor in thesame preparation using an [3H]-NαMH binding assay. As shown inTable 3 and Fig. 4, haloperidol has high affinity for the sigma-1 receptorand S 38093-2 showed approximately 30-fold selectivity for sigma-1receptors as compared to histamine H3 receptors. In contrast,GSK189254 and JNJ-5207852 displayed over 100-fold selectivity forthe histamine H3 receptor over the sigma-1 receptor. ABT-239, PF-3654746 and pitolisant displayed approximately equal affinity for bothreceptors (Table 3, Fig. 4).

3.4. S 38093-2 occupies sigma-1, but not histamine H3 receptors, at pro-cognitive doses in mice

In order to ascertain whether the affinity for sigma-1 receptors (andapparent selectivity over histamine H3 receptors) would be apparent atdoses of S 38093-2 associated with pro-cognitive activity (e.g.0.3–3 mg/kg in a contextual fear conditioning paradigm (Guillouxet al., 2017), and 0.1–3 mg/kg in a contextual memory task (Sors et al.,2016),), we performed (i) combined pharmacokinetic and ex vivo H3

and sigma-1 receptor occupancy study in mice, using GSK189254 as aselective H3 receptor antagonist control and (ii) complementary phar-macokinetic studies in rats to determine CSF levels of S 38093-2 free toaccess either H3 and sigma-1 receptors.

Total brain concentrations of S 38093-2 increased linearly after oraldoses over a range of 0.3–10 mg/kg in mice (15 min pre-treatment time;Fig. 5A). The measured brain concentrations of both S 38093-2 andGSK189254 (after a single dose of 1 mg/kg; 15 min pre-treatment time),when corrected for the degree of brain tissue binding (SupplementaryTable A2) to estimate the unbound fraction, were converted into %

histamine H3 or sigma-1 receptor occupancy using the affinity estimatesfrom Table 3 (Fig. 5B). These values suggest that S 38093-2 shouldoccupy approximately 40% of sigma-1 receptors after a dose of 0.3 mg/kg, and > 80% at 3 mg/kg, yielding an estimated ROcc50 of 0.4 mg/kg(Fig. 5D). However, the predicted occupancy of histamine H3 receptorswas much lower for S 38093-2, with an estimated ROcc50 of 9.3 mg/kg(Fig. 5B and D). In contrast, GSK189254 (1 mg/kg) would be predictedto robustly occupy mouse brain histamine H3 receptors without binding

Table 3Histamine H3 and sigma-1 affinity values of H3 receptor antagonists as mea-sured by competition for [3H]eN-α-methyl-histamine and [3H]-(+)-pentazo-cine binding in mouse brain homogenates. Data are expressed as mean ± SEMfrom a fit to grouped data from n = 6–8 independent experiments. Statisticalsignificance was determined by Student's t-test with *P < 0.05, ***P < 0.001and ****P < 0.0001 deemed significant.

Compound Histamine H3 pKI Sigma-1 (σ-1) pKI

JNJ-5207852 9.56 ± 0.35 5.93 ± 0.51***PF-3654746 8.00 ± 0.44 7.66 ± 0.02GSK189254 8.78 ± 0.71 6.62 ± 0.18*ABT-239 7.80 ± 0.70 8.20 ± 0.20Pitolisant 7.66 ± 0.02 8.00 ± 0.44S 38093-2 5.93 ± 0.51* 7.44 ± 0.26Haloperidol 5.15 ± 0.33 8.48 ± 0.17****

Fig. 4. Ex vivo compound affinities at the histamine H3R and sigma-1 re-ceptor. [3H]-(+)-Pentazocine (closed circles) and [3H]eN-α-methyl-histamine(NαMH; open circles) competition binding in mouse brain homogenates withthe H3 receptor antagonists JNJ-5207852, PF-3654746, GSK189254, ABT-239,pitolisant, S 38093-2, and the sigma-1 antagonist, haloperidol. Frozen wholeSwiss mouse brain was rapidly defrosted and homogenised in 55 mL w/v assaybinding buffer; homogenates (2 mg/well) were incubated with assay buffer orvarying concentration of test ligand. Non-specific binding (NSB) was de-termined by 10 μM haloperidol ([3H]-(+)-pentazocine binding) or 10 μM ABT-239 ([3H]-NαMH binding). Grouped data are shown ± SEM (n = 6–8).

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to sigma-1 receptors (Fig. 5B and D).Sigma-1 and H3 receptor occupancies for S 38093-2 and GSK189254

are shown in Fig. 5C and E. Although the absolute levels of occupancywere lower than predicted, it is clear that S 38093-2 occupies sigma-1receptors at doses of 0.3–1 mg/kg where there is little appreciablebinding to the histamine H3 receptor. Conversely, GSK189254 appearsto occupy only the histamine H3 receptor, with little binding to thesigma-1 receptor. Analysis of the dose-dependency yielded ROcc50 va-lues of 4.8 mg/kg (sigma-1) and 42 mg/kg (histamine H3) for S 38093-2. Although these values are 5-to-10-fold lower than the predicted va-lues, this is almost certainly due to ligand dissociation induced by di-lution of the tissue during the assay preparation (and possibly theaberrant assumption that the free ligand concentrations are at equili-brium with the target receptor). Complementary in vivo pharmacoki-netic studies in rats to determine the CSF levels of S 38093-2 after oraladministration 0.3 or 3 mg/kg (Fig. 6A) revealed drug levels that wouldbe predicted to robustly occupy sigma-1 receptors (∼20% at 0.3 mg/kg; > 90% at 3 mg/kg), with only approximately 40% H3 receptor oc-cupancy at the higher dose (Fig. 6B). These predictions correspond toestimated ROcc50 values of 0.65 and 4.5 mg/kg for sigma-1 and H3

receptor populations, respectively (Fig. 6C), which are close to thepredicted values for the mouse brain study described above.

Taken together, the predicted and/or measured values in bothmouse and rat confirm that S 38093-2 appears to preferentially occupythe sigma-1 receptor at low doses, suggesting that this pharmacologycould play a significant role in its in vivo activity.

4. Discussion

Several histamine H3 receptor antagonists have progressed intoclinical development for indications including Alzheimer's disease,schizophrenia, pain and narcolepsy. Each of the compounds studiedherein has been extensively characterized in its own right (Barbieret al., 2004; Esbenshade et al., 2005; Fox et al., 2005; Giannoni et al.,2010; Sors et al., 2017; Medhurst et al., 2008), but few studies havecompared their pharmacology in detail to determine common or dis-criminating properties that might be associated with in vivo efficacy.

Using functional cAMP and GTPγS assays we confirmed that all ofthe investigational and approved histamine H3 receptor antagonists are

Fig. 5. Combined pharmacokineticand ex vivo receptor occupancy studyin mice. Total brain concentrations of(A) S 38093-2 (0.3–10 mg/kg) andGSK189524 (1 mg/kg) after 15 min pre-treatment corrected for brain tissuebinding (Supplementary Table A2) toestimate the unbound fraction. In (B)estimated % histamine H3 or sigma-1receptor occupancy using the affinityestimates from Table 3, yielding esti-mated ROcc50 values (D). Sigma-1 andH3 receptor occupancies for S 38093-2and GSK189254 were determined asdescribed in the Methods and plotted as(C) % histamine H3 or sigma-1 receptoroccupancy to enable measured ROcc50

values (E). Grouped data are shown ±SEM (n = 6).

Fig. 6. CSF levels and predicted central sigma-1 and H3 receptor occu-pancy for S 38093–2 in rat. Time course of CSF levels of S 38093-2 after oraladministration (0.3 and 3 mg/kg) in Wistar rats. (B) Time course of predicted %histamine H3 or sigma-1 receptor occupancy using the affinity estimates fromTable 3, yielding approximate ROcc50 values at 30 min post-dose (C). Groupeddata are shown ± SEM (n = 3).

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inverse agonists of the receptor, displaying a broad range of affinities.There were no major discrepancies when comparing the affinities forthe full length, H3-445 receptor and the major truncated isoform, H3-365; although all the showed the same trend to have lower affinity forthe truncated isoform. This would be consistent with a higher level ofconstitutive receptor activation associated with this isoform in re-combinant cells (Bongers et al., 2007) and the confirmed inverse ago-nist activity of the compounds (Fig. 2), assuming a two-state model ofreceptor activation (viz. Canals et al., 2012).

Kinetic analysis of non-equilibrium Ca2+ mobilization assays re-vealed two major classes of H3 receptor antagonist: slowly dissociating(viz. GSK189254, PF-3654746) and fast dissociating (viz. ABT-239, pi-tolisant, S 38093-2). JNJ-5207852 displayed an interesting profile,appearing pseudo-irreversible at the full-length H3-445 receptor, buthaving faster dissociation kinetics at the shorter H3-365 isoform. It wasnoteworthy that dissociation rates were not well correlated with equi-librium dissociation constants; pitolisant and PF-3654746 had similarpKB values but very different dissociation rates (Table 2). This dis-crepancy is at odds with long-held dogma that dissociation rate drivesaffinity, although our data are consistent with other recent studies ofGPCR ligand kinetics that reveal that association rate may also varymarkedly and contribute to variance in observed equilibrium affinity(Riddy et al., 2015; Mould et al., 2014).

Whilst long receptor residence is often regarded as beneficial forantagonists (potentially improving duration of action and/or efficacy;Sykes et al., 2016; Seow et al., 2016; Strasser et al., 2017; Schuetz et al.,2017), a short receptor half-life apparently does not prevent pitolisantfrom exerting clinical benefit and of the compounds evaluated it is theonly one to be successful in Phase III and progress on to the market (asWakix, for narcolepsy; Syed, 2016; Calik, 2017). Likewise, rapidlydissociating ABT-239 (rapidly dissociating) appears to have betterwakefulness-promoting efficacy for the same degree of histamine H3

receptor occupancy than slowly dissociating GSK189254 (Le et al.,2008).

However, the overall pattern of histamine H3 receptor pharma-cology reported here is broadly consistent with the reported in vivoefficacy of the same compounds (Bitner et al., 2011; Medhurst et al.,2007; Barbier et al., 2004), with one significant exception. Unlike theother agents that display affinity in the nanomolar or sub-nanomolarrange (Tables 2 and 3), S 38093-2 is a rapidly dissociating, low-affinityhistamine H3 receptor antagonist. Although this is consistent withprevious reports (Sors et al., 2017), it is at odds with its in vivo

pharmacology. S 38093-2 displays potent activity in rodent models oflearning and memory, as well as neuropathic pain (Chaumette et al.,2018), with effective doses as low as 0.1–0.3 mg/kg (PO), whereas formarkers more classically linked to histamine H3 receptor blockade(such as pre-frontal cortical histamine or acetylcholine release andwakefulness in rats), effects are only seen at doses of 10 mg/kg (IP;Panayi et al., 2017).

Although H3 antagonist-induced wakefulness has been suggested torequire a higher degree of receptor occupancy than pro-cognitive ef-fects (Le et al., 2008), the 100-fold difference in effective doses led us toinvestigate whether other targets may be involved in the learning andmemory effects. Pan-receptor profiling revealed that S 38093-2 hasmicromolar antagonist activity in a guinea-pig vas deferens assay ofsigma receptor function, although when tested in an assay specificallytargeted at the central sigma-1 receptor (mouse brain [3H]-(+)-pen-tazocine binding), S 38093-2 displayed significantly (30-fold) higheraffinity for this target than for the histamine H3 receptor (Table 3).Indeed, when tested in an ex vivo mouse brain receptor occupancystudy, S 38093-2 preferentially occupied the sigma-1 receptor popula-tion compared to histamine H3 receptors.

The measured ROcc50 values (4.8 and 42 mg/kg, respectively) werelower than those estimated from the mouse free brain concentrations(0.4 and 9.3 mg/kg for the sigma-1 and histamine H3 receptor, re-spectively) or rat CSF levels (0.65 and 4.5 mg/kg, respectively). Thisdiscrepancy is almost certainly due to ligand dissociation during thesample preparation for the radioligand binding assay. For example, themeasured occupancy of H3 receptors in the rat cortex by the nanomolaraffinity antagonist, GSK189254, was approximately 4-fold lower thanwould be predicted by unbound brain levels (Supplementary FigureA2). It is therefore unsurprising that lower affinity and more rapidlydissociating ligands such as S 38093-2 would be even more prone todiscrepancies induced by tissue dilution, despite measures to minimizesuch effects.

Nonetheless, taken together the data suggest that there is likely tobe minimal histamine H3 receptor occupancy by S 38093-2 at dosesassociated with pro-cognitive and anti-hyperalgesic/anti-allodynic ac-tivity and that sigma-1 receptor occupancy could contribute to theseeffects.

The sigma-1 receptor is an unusual and poorly understood target,though it is thought to act as an intracellular chaperone (Hayashi et al.,2011) and is implicated in learning and memory, depression, anxietyand schizophrenia (Cobos et al., 2008; Zamanillo et al., 2013). Sigma-1

Fig. 7. Schematic diagram of the mechanisms of action of histamine H3R ligands including their histamine H3R dissociation kinetics and off-target sigma-1activity.

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antagonists are also effective in neuropathic pain (Abadias et al., 2013;Zamanillo et al., 2013) and, whilst the mechanism of action is not wellunderstood, several studies suggest sigma-1 receptors regulate ionchannel function (including NMDA/GluN receptors and K+/Ca2+

channels; Cobos et al., 2008).Binding to sigma-1 receptors may also play a role in the pharma-

cology of the other H3 receptor antagonists examined in this study.Whilst GSK189254 and JNJ-5207852 were highly selective for H3

versus the sigma-1 receptor, ABT-239, PF-2654746 and the approvedagent, pitolisant, all displayed similar affinity at both receptors. This isnot unprecedented for histamine H3 receptor antagonists: thioperamideand iodophenpropit were both shown to possess sub-micromolar affi-nity for sigma-1 receptor binding sites (Leurs et al., 1995). As such,sigma-1 affinity should be considered when interpreting the in vivo ef-ficacy of some of these agents.

Unfortunately, a lack of canonical functional assays for sigma re-ceptors makes classifying the pharmacology of sigma-targeting ligandschallenging – it is possible that differential agonist and antagonist ef-fects could potentially underlie some of the variations in pre-clinicaland clinical efficacy with H3 receptor antagonists. Sigma-1 receptorknockout mice or indirect assays, such as regulation of NMDA currentsvia SK channels in hippocampal slices (Martina et al., 2007) could helpunderstand sigma-1-mediated pharmacology. Interestingly, recent stu-dies have shown that the histamine H3 receptor and the sigma-1 re-ceptor can form heterodimers in both transfected cells and in rodentstriatum, and these may play a role in cocaine-seeking (Moreno et al.,2014). Such an interaction provides a potential physical mechanism bywhich the properties of S 38093-2 (herein and elsewhere; Panayi et al.,2017) may be reconciled. Recently an X-ray crystal structure of thesigma-1 receptor was solved, revealing it to be a single transmembranedomain protein (Schmidt et al., 2016); hopefully this will lead to fur-ther understanding of the function of this enigmatic protein family andthe drugs that target it.

In summary we demonstrate that S 38093-2 preferentially occupiessigma-1 receptors at pro-cognitive and anti-hyperalgesic/anti-allodynicdoses, whilst occupying the histamine H3 receptor at doses associatedwith wakefulness and neurotransmitter release. These findings helpexplain the in vivo pharmacology of S 38093-2 and aid in the differ-entiation of histamine H3 receptor antagonists, which in turn couldcontribute to some of their pre-clinical or clinical efficacy.

5. Conclusion

Our data allow pharmacological discrimination of diverse of H3

receptor antagonists that can be linked to their differential efficacy inpreclinical and clinical disease settings (Fig. 6). Moreover, we identifythe sigma-1 receptor as a common “off target” for H3 antagonists, andthat differential H3 and sigma-1 receptor occupancy may contribute toparadoxical efficacy of compounds such as S3803-2. The work providesan elaborated discovery path for understanding new candidate H3 re-ceptor antagonists.

Acknowledgements

A.C. is a Senior Principal Research Fellow and P.M.S. is a PrincipalResearch Fellow of the National Health and Medical Research Council(NHMRC) of Australia.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.neuropharm.2018.10.028.

Funding

This work was carried out and funded as part of a collaboration

between Monash University and Servier. Servier employees assistedwith the experimental design, interpretation of results and drafting ofthe manuscript.

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