ctep, a novel, potent, long acting, and orally bioavailable mglur5 inhibitor

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CTEP: A Novel, Potent, Long-Acting, and Orally Bioavailable Metabotropic Glutamate Receptor 5 Inhibitor S Lothar Lindemann, Georg Jaeschke, Aubin Michalon, Eric Vieira, Michael Honer, Will Spooren, Richard Porter, Thomas Hartung, Sabine Kolczewski, Bernd Bu ¨ ttelmann, Christophe Flament, Catherine Diener, Christophe Fischer, Silvia Gatti, Eric P. Prinssen, Neil Parrott, Gerhard Hoffmann, and Joseph G. Wettstein Pharmaceuticals Division, Discovery Neuroscience (L.L., A.M., M.H., W.S., R.P., C.D., C.Fi., S.G., E.P.P., J.G.W.), Discovery Chemistry (G.J., E.V., T.H., S.K., B.B.), Nonclinical Safety (C.Fl., N.P., G.H.), and Academic Alliances (R.P.), F. Hoffmann-La Roche Ltd., Basel, Switzerland Received July 4, 2011; accepted August 15, 2011 ABSTRACT The metabotropic glutamate receptor 5 (mGlu5) is a glutamate- activated class C G protein-coupled receptor widely expressed in the central nervous system and clinically investigated as a drug target for a range of indications, including depression, Parkinson’s disease, and fragile X syndrome. Here, we present the novel potent, selective, and orally bioavailable mGlu5 neg- ative allosteric modulator with inverse agonist properties 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H- imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 with low nanomolar affinity and shows 1000-fold selectivity when tested against 103 targets, including all known mGlu receptors. CTEP penetrates the brain with a brain/plasma ratio of 2.6 and displaces the tracer [ 3 H]3-(6-methyl-pyridin-2-ylethynyl)- cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice from brain regions expressing mGlu5 with an average ED 50 equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor is active in the stress-induced hy- perthermia procedure in mice and the Vogel conflict drinking test in rats with minimal effective doses of 0.1 and 0.3 mg/kg, respectively, reflecting a 30- to 100-fold higher in vivo potency compared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) and fenobam. CTEP is the first reported mGlu5 inhibitor with both long half-life of approximately 18 h and high oral bioavailability allowing chronic treatment with continuous receptor blockade with one dose every 48 h in adult and newborn animals. By enabling long-term treatment through a wide age range, CTEP allows the exploration of the full therapeutic potential of mGlu5 inhibitors for indications requiring chronic receptor inhibition. Introduction Glutamate is the predominant excitatory neurotransmitter in the central nervous system (CNS) involved in vital brain functions such as cognition, learning and memory, processing of emotions, and motor control. Glutamatergic neurotransmission is mediated by two types of receptors: 1) the ionotropic gluta- mate receptors, which are nonselective cation channels subdi- vided by their respective agonists into N-methyl-D-aspartate All financial support for this work has been provided by F. Hoffmann-La Roche Ltd. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.111.185660. S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: CNS, central nervous system; CTEP, 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine; DAT, dopamine transporter; GABA, -aminobutyric acid; GTPS, guanosine 5-O-(3-thio)triphosphate; CHO, Chinese hamster ovary; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography; IP, inositol phosphate; L-AP4, (2S)-2-amino-4-phosphonobutanoic acid; mGlu, metabotropic glutamate; MPEP, 2-methyl-6-(phenylethynyl)pyridine; MS, mass spectrometry; MTEP, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine; NET, norepinephrine transporter; NMDA, N-methyl-D-aspartate; NK, neurokinin; SERT, serotonin transporter; SIH, stress-induced hyperthermia; B/P, brain/ plasma; DMSO, dimethyl sulfoxide; ABP688, 3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime; LY341495, 2-[(1S,2S)-2- carboxycyclopropyl]-3-(9H-xanthen-9-yl)-D-alanine; SR142801, (S)-N-(1-{3-[(3R)-1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]propyl}-4- phenylpiperidin-4-yl-N-methylacetamide; WIN35,428, 2-carbomethoxy-3-(4-fluorophenyl)tropane; SCH 23390, R-( )-7-chloro-8-hydroxy-3-methyl-1- phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SR48968, N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide; G418, (2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-methylaminooxan-2-yl]oxy-2- hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol; LY354740, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; SIB-1757, 6-methyl-2- (phenylazo)-3-pyridinol; SIB-1893, ( E)-2-methyl-6-(2-phenylethenyl)pyridine. 0022-3565/11/3392-474–486$25.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 339, No. 2 Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 185660/3725466 JPET 339:474–486, 2011 Printed in U.S.A. 474 by guest on December 2, 2012 jpet.aspetjournals.org Downloaded from DC1.html http://jpet.aspetjournals.org/content/suppl/2011/08/17/jpet.111.185660. 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Page 1: CTEP, a novel, potent, long acting, and orally bioavailable mGluR5 inhibitor

CTEP: A Novel, Potent, Long-Acting, and Orally BioavailableMetabotropic Glutamate Receptor 5 Inhibitor□S

Lothar Lindemann, Georg Jaeschke, Aubin Michalon, Eric Vieira, Michael Honer,Will Spooren, Richard Porter, Thomas Hartung, Sabine Kolczewski, Bernd Buttelmann,Christophe Flament, Catherine Diener, Christophe Fischer, Silvia Gatti, Eric P. Prinssen,Neil Parrott, Gerhard Hoffmann, and Joseph G. WettsteinPharmaceuticals Division, Discovery Neuroscience (L.L., A.M., M.H., W.S., R.P., C.D., C.Fi., S.G., E.P.P., J.G.W.), DiscoveryChemistry (G.J., E.V., T.H., S.K., B.B.), Nonclinical Safety (C.Fl., N.P., G.H.), and Academic Alliances (R.P.), F. Hoffmann-LaRoche Ltd., Basel, Switzerland

Received July 4, 2011; accepted August 15, 2011

ABSTRACTThe metabotropic glutamate receptor 5 (mGlu5) is a glutamate-activated class C G protein-coupled receptor widely expressedin the central nervous system and clinically investigated as adrug target for a range of indications, including depression,Parkinson’s disease, and fragile X syndrome. Here, we presentthe novel potent, selective, and orally bioavailable mGlu5 neg-ative allosteric modulator with inverse agonist properties2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 withlow nanomolar affinity and shows �1000-fold selectivity whentested against 103 targets, including all known mGlu receptors.CTEP penetrates the brain with a brain/plasma ratio of 2.6 anddisplaces the tracer [3H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice

from brain regions expressing mGlu5 with an average ED50equivalent to a drug concentration of 77.5 ng/g in brain tissue.This novel mGlu5 inhibitor is active in the stress-induced hy-perthermia procedure in mice and the Vogel conflict drinkingtest in rats with minimal effective doses of 0.1 and 0.3 mg/kg,respectively, reflecting a 30- to 100-fold higher in vivo potencycompared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) andfenobam. CTEP is the first reported mGlu5 inhibitor with bothlong half-life of approximately 18 h and high oral bioavailabilityallowing chronic treatment with continuous receptor blockadewith one dose every 48 h in adult and newborn animals. Byenabling long-term treatment through a wide age range, CTEPallows the exploration of the full therapeutic potential of mGlu5inhibitors for indications requiring chronic receptor inhibition.

IntroductionGlutamate is the predominant excitatory neurotransmitter

in the central nervous system (CNS) involved in vital brainfunctions such as cognition, learning and memory, processing ofemotions, and motor control. Glutamatergic neurotransmissionis mediated by two types of receptors: 1) the ionotropic gluta-mate receptors, which are nonselective cation channels subdi-vided by their respective agonists into N-methyl-D-aspartate

All financial support for this work has been provided by F. Hoffmann-LaRoche Ltd.

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

doi:10.1124/jpet.111.185660.□S The online version of this article (available at http://jpet.aspetjournals.org)

contains supplemental material.

ABBREVIATIONS: CNS, central nervous system; CTEP, 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine;DAT, dopamine transporter; GABA, �-aminobutyric acid; GTP�S, guanosine 5�-O-(3-thio)triphosphate; CHO, Chinese hamster ovary; HEK, humanembryonic kidney; HPLC, high-performance liquid chromatography; IP, inositol phosphate; L-AP4, (2S)-2-amino-4-phosphonobutanoic acid; mGlu,metabotropic glutamate; MPEP, 2-methyl-6-(phenylethynyl)pyridine; MS, mass spectrometry; MTEP, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine; NET,norepinephrine transporter; NMDA, N-methyl-D-aspartate; NK, neurokinin; SERT, serotonin transporter; SIH, stress-induced hyperthermia; B/P, brain/plasma; DMSO, dimethyl sulfoxide; ABP688, 3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime; LY341495, 2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-D-alanine; SR142801, (S)-N-(1-{3-[(3R)-1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]propyl}-4-phenylpiperidin-4-yl-N-methylacetamide; WIN35,428, 2�-carbomethoxy-3�-(4-fluorophenyl)tropane; SCH 23390, R-(�)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SR48968, N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide;G418, (2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-methylaminooxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol; LY354740, (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid; SIB-1757, 6-methyl-2-(phenylazo)-3-pyridinol; SIB-1893, (E)-2-methyl-6-(2-phenylethenyl)pyridine.

0022-3565/11/3392-474–486$25.00THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 339, No. 2Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics 185660/3725466JPET 339:474–486, 2011 Printed in U.S.A.

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(NMDA), �-amino-3-hydroxy-5-methy-4-isoxazoleproprionic acid(AMPA), and 2-carboxyl-3-carboxymethyl-4-isopropenylpyrroli-dine (kainate) receptors; and 2) the metabotropic glutamate(mGlu) receptors 1 to 8, which are G protein-coupled receptorsmodulating synaptic function (Kew and Kemp, 2005). ThemGlu5 receptor is a class C G protein-coupled receptor couplingto Gq-type G proteins, and its signal transduction is mediatedmainly by the activation of phospholipase C�, intracellular re-lease of inositol phosphates and Ca2� and the activation ofprotein kinase C and mitogen-activated kinase. In the nativetissue context, numerous protein-protein interactions withother receptors, scaffolding, and cytoplasmic proteins add com-plexity to the signal transduction of mGlu5 (Bockaert et al.,2010; Ribeiro et al., 2010). In the CNS the mGlu5 receptor isexpressed in the limbic cortex, hippocampus, amygdala, thala-mus, olfactory tubercle, and basal ganglia (Spooren et al., 2003).In neurons, mGlu5 is found primarily postsynaptically in excit-atory terminals often colocalized with adenosine A2a, dopamineD2, and NMDA receptors. In addition, mGlu5 is expressed inastrocytes where its function is not yet fully resolved (Hamiltonand Attwell, 2010).

The pharmacological toolset for investigating mGlu5 recep-tor function was initially limited to amino acid-like or-thosteric agonists and antagonists with unsatisfactory selec-tivity, potency, and brain penetration, which were restrictedprimarily to in vitro use (Kew and Kemp, 2005). The discov-ery of potent and systemically active mGlu5-negative alloste-ric modulators such as 2-methyl-6-(phenylethynyl)pyridine(MPEP) (Gasparini et al., 1999), 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (MTEP) (Cosford et al., 2003), and fenobam(N-(3-chlorophenyl)-N�-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea (Porter et al., 2005) triggered intenseresearch addressing the therapeutic potential of mGlu5. Pre-clinical studies largely generated with these prototypicalmGlu5 inhibitors support mGlu5 as a drug target and havetranslated into drug development programs in a wide rangeof indications including anxiety and depression, Parkinson’sdisease, pain, addiction, Huntington’s disease, and fragile Xsyndrome (Gasparini et al., 2008; for review, see Jaeschke etal., 2008). The prototypical mGlu5 inhibitors are invaluableresearch tools, which, however, have limitations for in vivouse because of their low metabolic stability translating intoshort half-lives. Especially for chronic in vivo experimentsrequiring sustained receptor blockade, the need for severaldaily drug administrations presents logistical challenges, canconfound phenotypes of animal models and behavioral tests,and is not feasible, e.g., when working with newborn animalsor fragile transgenic mouse lines. The in vivo use of fenobamis limited by an extensive metabolism (Wu et al., 1995) lead-ing to variable exposure and possibly producing pharmaco-logically active metabolites (Porter et al., 2005). For MPEP, aweak inhibition of the norepinephrine transporter (Heidbre-der et al., 2003), weak activities on NR2B-containing NMDAreceptors and monoamine oxidase A (Lea and Faden, 2006),and positive allosteric modulation of mGlu4 (Mathiesen etal., 2003) have been reported.

Here is described the pharmacological profile of the novelpotent, selective, and orally bioavailable mGlu5 negative allo-steric modulator with inverse agonist activity 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine) (CTEP). The new mGlu5 inhibitor binds hu-man, mouse, and rat mGlu5 with high affinity and low species

difference (Kd � 1.7, 1.8, and 1.5 nM for human, mouse, and ratmGlu5, respectively). The high affinity translates into 30- to100-fold higher in vivo potency compared with MPEP and feno-bam in two rodent behavioral models sensitive to antianxietydrugs. CTEP is �1000-fold selective for mGlu5 when tested on103 molecular targets, including all known mGlu receptors. Theunique feature of CTEP is its pharmacokinetic profile charac-terized by a brain/plasma (B/P) ratio of 2.6 and a high oralbioavailability of approximately 100%. The compound can bedosed orally, intraperitoneally, or subcutaneously formulated ina simple microsuspension. With its long half-life of approxi-mately 18 h in mice, CTEP enables chronic dosing with sus-tained receptor blockade with one dose every 48 h.

Materials and MethodsMaterials

CTEP (RO4956371), MPEP, MTEP, and fenobam were synthesizedat F. Hoffmann-La Roche Ltd. The tracer [3H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) was synthe-sized essentially as described previously (Hintermann et al., 2007),[3H]CTEP, [3H]flumazenil, and [3H] 2-[(1S,2S)-2-carboxycyclopropyl]-3-(9H-xanthen-9-yl)-D-alanine (LY341495) were synthesized at F. Hoff-mann-La Roche Ltd. All other radiolabeled compounds were purchasedfrom Anawa Trading SA (Wangen, Switzerland; [3H]MPEP), GEHealthcare [Glattbrug, Switzerland; [3H]N-(1-{3-[(3R)-1-benzoyl-3-(3,4-dichlorophenyl)piperidin-3-yl]propyl}-4-phenylpiperidin-4-yl-N-methyl-acetamide (SR142801)], and PerkinElmer (Schwerzenbach, Switzer-land; [3H]citalopram, [3H]8-cyclopentyl-1,3-dipropylxanthine, [35S]GTP�S, 125I-5�-N-methylcarboxamidoadenosine, [3H]nisoxetine,[3H]pyrilamine, [3H]2�-carbomethoxy-3�-(4�-fluorophenyl)tropane(WIN35,428), [3H]R-(�)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH 23390), [3H]N-[(2S)-4-(4-acetamido-4-phenylpiperidin-1-yl)-2-(3,4-dichlorophenyl)butyl]-N-methylbenzamide (SR48968), [3H]SR142801, and 125I-Bolton Hunter-substance P). All other drugs, chemicals, and enzymes were purchasedat the highest purity available from Tocris Bioscience (Bristol, UK),Sigma Chemie (Buchs, Switzerland), Roche Applied Sciences (Rot-kreuz, Switzerland), GE Healthcare, and Thermo Fischer Scientific(Wohlen, Switzerland). Cell culture reagents were purchased from In-vitrogen (Basel, Switzerland), cell culture plastic ware was purchasedfrom Nunc (Langenselbold, Germany), and all other plastic ware andconsumables were purchased from BD Biosciences (Allschwil, Switzer-land), Eppendorf AG (Basel, Switzerland), Greiner Bio-One GmbH(Frickenhausen, Germany), Hamamatsu Corporation (Solothurn, Swit-zerland), Rainin Instruments (Greifensee, Switzerland), and Semadeni(Ostermundigen, Switzerland).

Plasmids, Cell Culture, and Membrane Preparations

For in vitro pharmacological assays, plasmids encoding (Genbankaccession number and vectors in brackets) human adenosine A1 recep-tor [NM_000674; pCDNA3.1(�)], human adenosine A3 receptor[L22607; pCDNA3.1(�)], human dopamine D1 receptor [NM_000794;pCDNA3.1(�)], human GABAA subunits �5 [NM_000810; pIRES-NEO2], �3 [NM_000814; pIRES-PURO2], and �2 [NM_000816; pIRES-HYGRO2], human histamine H1 receptor [NM_000861; pCDNA3,1(�)],human mGlu1a [NM_000838; pcDNA5/FRT/TO], human mGlu2[NM_000839; pcDNA5/FRT/TO], human mGlu3 [NM_000840;pcDNA5/FRT/TO], rat mGlu4a [NM_022666; pSFV2.gen], humanmGlu5a [D28538; pcDNA5/FRT/TO], mouse mGlu5a [NM_001081414;pCDNA3.1(�)], rat mGlu5a [NM_017012; pcDNA5/FRT/TO], ratmGlu6 [NM_022920.1; pCDNA3.1(�)], human mGlu7a [NM_000844.3;pCDNA3.1(�)], human mGlu8 [NM_000845.2; pcDNA5/FRT/TO], hu-man serotonin transporter (SERT) [NM_001045; pcDNA3], human nor-epinephrine transporter (NET) [NM_001172502; pcDNA3], human do-pamine transporter (DAT) [NM_001044; pcDNA3], human neurokinin

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receptors 1 to 3 (NK1–3) [NK1, NM_001058, pCI-Neo; NK2, NM_001057,pCI-Neo; NK3, P29371, pCI-Neo], mouse G�15 [M80632; pIRES-NEO2], human G�16 [M63904; pIRES-NEO2], and rat excitatoryamino acid transporter 1 [U39555; pCDNA3.1(�)] were used.

For in vitro radioligand binding assays, membranes isolated fromHEK293 cells transiently (A1, A3, D1, mGlu3, mGlu5a, SERT, NET,DAT, and NK1–3) or stably (GABAA �5�3�2, SERT, NET, and DAT)transfected with plasmids encoding the respective receptors or re-ceptor subunit combinations (GABAA and �5�3�2) were used. Cellculture and transfections were performed essentially as describedpreviously (Porter et al., 2005;). Cell membranes were prepared fromcells harvested 48 h after transfection. The cells were washed threetimes in cold phosphate-buffered saline, suspended in cold homoge-nization buffer (50 mM Tris-HCl and 10 mM EDTA, pH 7.4), andhomogenized with a polytron (Kinematica AG, Basel, Switzerland)for 10 s at 10,000 rpm. The pellet obtained after centrifugation for 30min at 48,000g at 4°C was resuspended in homogenization buffercontaining 10 mM Tris-HCl and 0.1 mM EDTA, homogenized, andcentrifuged as above, and the resulting pellet was resuspended in asmall volume of homogenization buffer. The protein content of themembrane suspension was determined with the Bradford method(Bio-Rad Laboratories, Reinach, Switzerland) using �-globulin asstandard, and aliquots of the membrane preparation were stored at�80°C. Membranes from cortex tissue dissected from Wistar ratbrain were prepared following essentially the same procedure asdescribed for cells.

In Vitro Pharmacology

Radioligand Binding. For all filtration radioligand binding as-says, membrane preparations expressing the target receptors orreceptor combinations were resuspended in radioligand binding buf-fer (15 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 1.25 mM CaCl2, and1.25 mM MgCl2, pH 7.4), and the membrane suspension was mixedwith the appropriate concentrations of radioligand and nonlabeleddrugs in 96-well plates in a total volume of 200 l and incubated for60 min at the appropriate temperature. At the end of the incubation,membranes were filtered onto Whatman (Clifton, NJ) Unifilter (96-well microplate with bonded GF/C filters) preincubated with 0.1%polyethyleneimine in wash buffer (50 mM Tris-HCl, pH 7.4) with aFiltermate 196 harvester (PerkinElmer) and washed three timeswith ice-cold wash buffer. Radioactivity captured on the filter wasquantified on a Topcount microplate scintillation counter (Perkin-Elmer) with quenching correction after the addition of 45 l of Micro-Scint 40 per well and shaking for 20 min. The concentration ofmembranes and incubation time was determined for each assay inpilot experiments. Radioligand binding assays on SERT, NET, andDAT (Tatsumi et al., 1997; Liu et al., 2009) and the A1 adenosinereceptor were performed using the scintillation proximity assay prin-ciple (Bosworth and Towers, 1989). Selectivity screening at CEREP(Poitiers, France) (Table 2) was conducted at a concentration of 1 Min duplicate for each target except for kainate glutamate receptors,L-type Ca2� channel (dihydropyridine site), and Na2� channel,which were examined in a dose-response manner up to a concentra-tion of 10 M. Assay conditions for binding experiments are summa-rized in Supplemental Table S1.

For competition binding experiments the concentration of radioli-

gands was adjusted according to pilot experiments (SupplementalTable S1); nonspecific binding was measured in the presence of ahigh concentration (�100 Ki) of a known displacer of each radioli-gand. Typically, radioligand displacements were measured with 8 to10 concentrations of the test compounds. IC50 values were derivedfrom inhibition curves using XLfit software (ID Business Solutions,Guildford, UK), and Ki values were calculated according to the equa-tion Ki � IC50/(1 � [L]/[Kd]) (Cheng-Prusoff equation; Cheng andPrusoff, 1973), where [L] is the concentration and [Kd] the dissocia-tion constant of the radioligand. Saturation isotherms were deter-mined typically using 12 concentrations of the radioligand, essen-tially following the filtration radioligand binding procedure withoutantagonists described above. The Kd values were calculated usingXLfit software with a single-site specific binding rectangular hyper-bolic equation based on the law of mass action B � Bmax [F]/(Kd �[F]), where B is the amount of radioligand bound at equilibrium,Bmax is the number of available binding sites, [F] is the concen-tration of unbound radioligand, and Kd is the radioligand dissoci-ation constant.

Ca2� Mobilization Assays. For Ca2� mobilization assays,HEK293 cells stably (mGlu1a, mGlu2, human mGlu5a, rat mGlu5a,and mGlu8) or transiently (mouse mGlu5a) transfected with plas-mids encoding the respective target receptor or with a combination ofthe target receptor and G�15 (mGlu8) or G�16 (mGlu2) were used.Cells were seeded at 5 104 cells/well in poly-D-lysine-coated, 96-well, black/clear-bottomed plates. After 24 h, the cells were loadedfor 1 h at 37°C with 2.5 M Fluo-4 acetoxymethyl ester in loadingbuffer (1 Hanks’ balanced salt solution and 20 mM HEPES). Thecells were washed five times with loading buffer to remove excessdye, and intracellular calcium mobilization (intracellular calciumconcentration) was measured using a FDSS7000 device(Hamamatsu).

The potency of CTEP and, where available, reference antagonistswere studied in the presence of an agonist [mGlu1 and mGlu2,glutamate; mGlu5, quisqualate; mGlu8, (2S)-2-amino-4-phospho-nobutanoic acid (L-AP4)] at a concentration triggering 60 to 80% ofthe maximal agonist response, which was determined daily in aseparate experiment. The antagonists were applied in a serial dilu-tion with 10 different concentrations 30 min before the application ofagonists; potential agonist activities of CTEP were monitored on-lineduring the 5- to 30-min preincubation period (data not shown).Responses were measured as peak increase in fluorescence recordedafter the addition of CTEP and reference antagonists (testing foragonist activity), as well as after the addition of agonist (testing forantagonist activity), minus basal (i.e., fluorescence without additionof agonist), normalized to the maximal stimulatory effect induced bya saturating concentration of the agonist measured on the sameplate. Inhibition curves were fitted using XLfit according to the Hillequation y � 100/(1 � (x/ IC50)nH), where nH is the slope factor.

Inositol Phosphate Accumulation Assay. For inositol phos-phate (IP) accumulation assays, HEK293 cells stably (human and ratmGlu5) or transiently (mouse mGlu5) transfected with plasmidsencoding the respective target receptor were transfected with excit-atory amino acid transporter 1 and seeded in poly-D-lysine-coated96-well plates at a density of 80,000 cells/well in an inositol/gluta-mate-free Dulbecco’s Modified Eagle’s Medium with 10% dialyzedfetal bovine serum, 1% penicillin/streptomycin, 1 mM glutamate,and 5 Ci/ml myo-[2–3H]inositol. After 24 h, cells were washed threetimes with washing buffer containing 1 Hanks’ balanced salt solu-tion and 20 mM HEPES and incubated in the presence of glutamatepyruvate transaminase (Roche Applied Science, Indianapolis, IN)and sodium pyruvate for 1 h before the addition of agonists orantagonists in assay buffer (washing buffer containing 8 mM LiCl).After 45-min incubation with the agonist, the assay was terminatedby the aspiration of the assay buffer and the lysis of cells with theaddition of 100 l 20 mM formic acid per well. After 20-min incuba-tion, a 20-l aliquot of the lysate was mixed with 80 l of RNAbinding yttrium silicate bead suspension that bound to the inositol

TABLE 1Chromatography gradient for 2.5-min analytical runs

Time Solvent A Solvent B

%

0.01 min 10 900.50 min 30 701.00 min 95 51.80 min 95 51.90 min 95 52.00 min 10 90

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phosphates but not to inositol. The plates were counted on a Top-count microplate scintillation counter (PerkinElmer), and data wereexpressed as a percentage of the basal signal obtained in the absenceof drugs.

[35S]GTP�S Binding Assay. For [35S]GTP�S binding assays,membranes were isolated as described above from CHO cells trans-fected with semliki virus encoding mGlu4a and mGlu6, respectively.The membranes were mixed in a total volume of 180 l per well witha serial dilution of CTEP in 10 different concentrations, 0.3 M GDP,0.3 nM [35S]GTP�S, and L-AP4 at a concentration triggering 60 to80% of the maximal agonist response, and 1 mg/well wheat germagglutinin SPA beads. The plates were incubated for 30 min at roomtemperature under mild shaking, centrifuged for 3 min at 1000g, andcounted immediately in a Topcount microplate scintillation counter(PerkinElmer). Data subtracted with the background (reaction withno agonist or drug in presence of 10 M cold GTP�S) were normal-ized to the maximal stimulatory effect induced by a saturating con-centration of L-AP4 measured on the same plate.

TABLE 2Selectivity profile of CTEPData generated at a single concentration of 1 M CTEP represent the mean of n �2, Ki and IC50 values generated with a concentration range up to 10 M CTEPrepresent the mean of n � 2–6. Activity is expressed as percent control displacementfor single concentration measurements in radioligand binding experiments, as per-centage of control inhibition for single-concentration measurements in functionalassays or as Ki or IC50 for radioligand binding or functional assays recordingconcentration-response measurements, respectively.

Activity

% Control Ki/IC50

MRadioligand Binding AssaysReceptors: low molecular weight ligands

Adenosine A1 receptor (h)a 6.2Adenosine A2A receptor (h)b N.A.D.Adenosine A3 receptor (h)a 2.3Adrenergic �1 receptor (N.S.) (r)b 9Adrenergic �2 receptor (N.S.) (r)b 5Adrenergic �1 receptor (h)b 3Adrenergic �2 receptor (h)b 5Androgen receptor (h)b 5Cannabinoid receptor 1 (h)b 9Cannabinoid receptor 2 (h)b 11Dopamine D1 receptor (h)a �10Dopamine D2 receptor (h)b N.A.D.Dopamine D3 receptor (h)b 8Dopamine D4.4 receptor (h)b 7Estrogen receptor (N.S.) (h)b N.A.D.Glucocorticoid receptor (h)b N.A.D.Histamine receptor 1 (h)a �4Histamine receptor 2 (h)b N.A.D.Histamine receptor 3 (h)b 5Imidazolin receptor 1 (b)b 3Imidazolin receptor 2 (r)b 9Melanocortin receptor 4 (h)b 15Metabotropic glutamate receptor 2a �10Muscarinic receptors (N.S.) (r)a 5.3Opioid receptor (N.S.) (r)b N.A.D.Progesterone receptor (h)b N.A.D.Purinergic P2X receptor (r)b N.A.D.Purinergic P2Y receptor (r)b N.A.D.Serotonin receptor (N.S.) (r)b N.A.D.Sigma receptor (N.S.) (r)b 6

Receptors: peptides and lipidsAngiotensin receptor 1 (h)b 8Angiotensin receptor 2 (h)b N.A.D.Arginine vasopressin receptor 1a (h)b 5Arginine vasopressin receptor 2 (h)b N.A.D.Bradykinin receptor 1 (h)b 3Bradykinin receptor 2 (h)b 5Cholecystokinin receptor type A (h)b N.A.D.Cholecystokinin receptor type B (h)b N.A.D.Corticotropin-releasing factor receptor 1 (h)b 1Endothelin receptor type A (h)b N.A.D.Endothelin receptor type B (h)b N.A.D.Leukotriene B4 receptor (LTB4, BLT1) (h)b N.A.D.Leukotriene D4 receptor (LTD4, CysLT1) (h)b 16Neurokinin receptor 1 (h)a �10Neurokinin receptor 2 (h)a �10Neurokinin receptor 3 (h)a �10Neuropeptide Y receptor (N.S.) (r)b 7Nociceptin receptor (ORL1) (h)b 8Peroxisome proliferator-activated receptor � (h)b 3Prostanoid EP2 receptor (h)b 3Prostanoid IP receptor (h)b N.A.D.Thyrotropin releasing hormone receptor

(TRH1) (h)b12

TransporterNorepinephrine transporter (h)a �10Dopamine transporter (h)a �10GABA transporter (r)b 8Choline transporter (CHT1) (h)b N.A.D.Serotonin transporter (h)a �10

Ion channelsAcetylcholine receptor, nicotinic

(�4�2�-BGTX-insensitive) (r)bN.A.D.

AMPA glutamate receptor (r)b N.A.D.

TABLE 2—Continued

Activity

% Control Ki/IC50

MRadioligand Binding Assays

Ca2� channel, L-type (DHP site) (r)b 2.7Ca2� ch., L-type (diltiazem site) (r)b 15Ca2� ch., L-type (verapamil site) (r)b 11Ca2� channel, SK (N.S.) (r)b N.A.D.GABAA (N.S.) (r)b 16GABAA (central, BZD) (r)b 4GABAA (central, BZD; �5�3�2) (h)a �3.2Kainate glutamate receptor (r)b 7.9K� ch., ATP-sensitive (Kir 6.2) (r)b 2K� ch., voltage gated (�-DTX) (r)b N.A.D.Na� channel (site 2) (r)b 5NMDA glutamate receptor (PCP) (r)b 14NMDA glutamate receptor (r)b N.A.D.

Functional assaysAcetylcholinesterase (h)b 7Adenylate cyclase (r)b N.A.D.ATPase (Na�/K�) (p)b N.A.D.Catechol-O-methyltransferase (p)b N.A.D.GABA transaminase (r)b N.A.D.Guanylate cyclase (b)b N.A.D.Histone deacetylase (HDAC)3 (h)b N.A.D.Histone deacetylase (HDAC)4 (h)b 2Histone deacetylase (HDAC)6 (h)b N.A.D.Histone deacetylase (HDAC)11 (h)b N.A.D.Metabotropic glutamate receptor 1a �10Metabotropic glutamate receptor 3a �10Metabotropic glutamate receptor 4a �10Metabotropic glutamate receptor 6a �10Metabotropic glutamate receptor 7a �10Metabotropic glutamate receptor 8a �10Monoaminoxidase A (h)b 3Monoaminoxidase B (h)b 5Phenylethanolamine N-methyl

transferase (PNMT) (b)bN.A.D.

Phosphodiesterase 1 (b)b N.A.D.Phosphodiesterase 2 (h)b 4Phosphodiesterase 3 (h)b N.A.D.Phosphodiesterase 4 (h)b 5Phosphodiesterase 5 (h)b 9Phosphatase 1B (h)b 4Phosphatase CDC25A (h)b 5Phosphatase MKP1 (h)b 1Protein kinase C� (h)b N.A.D.Sirtuin 1 (h)b N.A.D.Sirtuin 2 (h)b 2Tyrosine hydroxylase (r)b N.A.D.

N.A.D., no activity detected; N.S., nonselective; h, human; r, rat; b, bovine; p,porcine.

a Data generated at F. Hoffmann-La Roche Ltd.b Data generated at CEREP.

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cAMP Accumulation Assay. For the mGlu7a cAMP accumulationassay, a clonal CHO cell line deficient in dihydrofolate reductase activ-ity (CHO-dhfr�), harboring a luciferase reporter gene under the controlof 5 cAMP-responsive elements (CHO-DUKX-CRE-luci cells) and stablytransfected with mGlu7a was used. Cells were seeded at 5 104

cells/well in poly-D-lysine coated, 96-well, black/clear-bottomed plates.After 24 h the growth medium [Dulbecco’s Modified Eagle’s Medium,7.5% heat-inactivated fetal bovine serum, 1 hypoxanthine/thymidinesupplement, 0.2 mg/ml hygromycin, 0.4 mg/ml (2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-[(2R,3R,4R,5R)-3,5-dihy-droxy-5-methyl-4-methylaminooxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol (G418), 1 mM L-glutamate, and 0.34mM L-proline] was replaced with 1 mM 3-isobutyl-1-methylxanthine inKrebs Ringer bicarbonate buffer, and the plates were incubated for 60min at 30°C. After adding CTEP in a serial dilution with nine differentconcentrations, plates were incubated for 15 min at 30°C, then forskolinand L-AP4 were added at final concentrations of 3 M and 3 mM,respectively, and plates were incubated for 30 min at 30°C. The cAMPdetection was performed with the cAMP-Nano-TRF detection assay(Roche Applied Science) according to the manufacturer’s instructions.Data subtracted with the background (reaction with no drug and noforskolin and L-AP4) were normalized to the maximal stimulatoryeffect induced by L-AP4 in the presence of 3 M forskolin.

Selectivity Testing at CEREP. CTEP’s selectivity for mGlu5was assessed at CEREP using the Diversity Profile panel at a con-centration of 1 M in duplicates. Targets showing an effects size of�20% control were retested in a dose-response fashion at CEREP orF. Hoffmann-La Roche Ltd. (Table 2). Key experimental conditionsused in the selectivity profiling of CTEP are summarized in Supple-mental Table S1.

Behavioral Pharmacology

Animals and Drug Treatment. All experiments with animalswere conducted in accordance with federal and local regulations andwith the approval of the City of Basel Animal Protection Committee.

Adult male Sprague-Dawley rats (body weight approximately180–210 g) and male NMRI mice (body weight approximately 25 g)were supplied by Charles River (Sulzfeld, Germany). Rats weregroup-housed and mice were-single housed in separate holdingrooms at controlled temperature (20–22°C) and 12-h light/dark cycle(lights on 6:00 AM). Animals were allowed ad libitum access to foodand water, with the exception of those used in the Vogel conflictdrinking test, where access to water was limited during the trainingsessions as described below. All formulations were prepared imme-diately before use in vehicle, consisting of 0.9% NaCl (w/v) and 0.3%Tween 80 (v/v) solution for oral administration of CTEP, MPEP,MTEP, and fenobam; 0.9% NaCl solution for MPEP and MTEPintravenously; and 30% N-methylpyrrolidone, 42% hydroxypropyl-�-cyclodextrin, and 28% water for fenobam intravenously. The volumeof administration for oral dosing was 5 ml/kg for rats, 10 ml/kg formice, and 2.5 ml/kg for intravenous applications and 10 ml/kg forsubcutaneous applications in mice.

Microsuspensions of CTEP, MPEP, MTEP, and fenobam wereprepared by milling the compounds in the appropriate vehicle in aplanetary ball mill (Retsch GmbH, Haan, Germany) with glass beadsfor 90 min. The resulting suspension was vacuum-filtered through a100-m filter, adjusted to the final concentration, and stored frozenin aliquots at �80°C before use.

SIH in Mice. The SIH test was essentially performed as describedpreviously (Spooren et al., 2002). In brief, drug-naive, single-housed(Makrolon cage, 26 21 14 cm) male NMRI mice were used, andbody temperature was measured twice in each mouse 1 min apart(T1 and T2, respectively). T1 served as the handling stressor, and theresulting increase in body temperature (�T � T2 � T1) reflected theSIH. Mice were administered drug or vehicle orally 60 min beforemeasuring T1.

Vogel Conflict Drinking in Rats. Male Sprague-Dawley ratswere water-restricted for three consecutive 24-h periods. At the end

of the first 24-h period, animals were allowed access to water in theirhome cage for 60 min. At the end of the second 24-h period, they wereplaced into the test chamber containing a drinking bottle and wereallowed to drink freely for 15 min. Subsequently they received drink-ing water in their home cage for another 60 min. The drinking timein the test chamber was used to randomize the animals over thedifferent treatments. The drinking time measured by an opticalsensor was determined as the time spent by the rat at the drinkingspout. At the end of the third 24-h period, rats were administered theexperimental compounds or vehicle orally and isolated in a holdingchamber until testing 60 min later. During the 10-min test period,they were allowed to drink freely for a cumulative time of 5 s, afterwhich drinking was associated with an electric stimulus. That is, anelectrical shock between the grid floor and the drinking spout (0.5mA; 250 ms) was triggered every second of cumulative drinking time.This shock level strongly suppresses drinking to approximately 10%of normal levels.

Statistical Analysis of Behavioral Data. Statistical analyseswere performed using Statistica (StatSoft, Hamburg, Germany). SIHdata were statistically evaluated using a Dunnett multiple compar-ison test (two-tailed) after one-way analysis of variance. For theVogel conflict drinking procedure, drinking time was analyzed byMann-Whitney U post hoc tests. In all experiments, the level ofsignificance was p � 0.05. For uniformity, all data are plotted asmean S.E.M. in the figures.

Pharmacokinetics, In Vivo Binding, and PhysicochemicalProperties

Pharmacokinetics. For blood sample collection from adult mice,two consecutive blood samples were collected from each animal. Thenumber of adult animals used per dose and route was typically fiveto seven (two analyzed samples for each data point). The recordedplasma profiles therefore represent composite profiles generatedfrom several animals per compound and administration route. Fornewborn mice, only brain tissue was analyzed (one pooled samplefrom three to four animals per time point). Brain/plasma ratios forMPEP, MTEP, and fenobam were analyzed 1 h after a single oraldose of 30 mg/kg in adult mice.

For preparation of plasma samples approximately 1 ml of bloodwas collected into EDTA-coated 1.5-ml Eppendorf tubes and centri-fuged for 5 min at 5000g, and the clear supernatant was transferredinto a clean tube. Before analysis, 50 l of plasma samples weremixed with 150 l of acetonitrile containing the internal standardbosentan (200 ng/ml) in a 96-deep-well plate for protein precipita-tion. After centrifugation at 5900g for 10 min at room temperature,100 l of supernatant was mixed with 100 l of water. From theabove plasma extracts, 10 l were subjected to HPLC/MS analysis.

Brain samples were homogenized in three volumes (w/v) of waterin a planetary ball mill (Retsch GmbH) with ceramic beads and thendiluted with one volume of brain homogenate from drug-naive ani-mals of the same species and age. From this homogenate 50 l weremixed with 150 l of acetonitrile containing the internal standardbosentan (200 ng/ml) for protein precipitation. After centrifugationat 5900g for 10 min at room temperature, 100 l of supernatant wasmixed with 100 l of water. From the above plasma extracts, 10 lwere subjected to HPLC/MS analysis.

Samples were analyzed using a combined HPLC/MS method.Standard solutions for the unknown compounds (CTEP, MPEP,MTEP, and fenobam), as well as the internal standard bosentan,were freshly prepared for each analysis. Solutions of unknown com-pounds in DMSO were serially diluted in DMSO to concentrations of12.5 to 125,000 ng/ml and mixed 1:50 with plasma and brain extractsderived from drug-naive animals. A stock solution of bosentan inDMSO at a concentration of 1 mg/ml was diluted 1:5 in methanol.

Chromatographic analyses were performed using a Phenomenex(Torrance, CA) Luna C18 (50 1.0 mm) 5.0-m, 100A analyticalcolumn at 70°C. The flow rate was 600 l/min. A 15-l mixing

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chamber was used to mix the mobile phase, which was a mixture ofsolvents A (10 mM ammonium acetate and 0.05% v/v formic acid inwater) and B (acetonitrile containing, 0.05% v/v formic acid). Thechromatography gradient for 2.5-min analytical runs is shown inTable 1.

Chromatography eluates were analyzed using a heated TurboIon-Spray mass spectrometer (AB SCIEX; Brugg, Switzerland). The gasused was nitrogen with the following settings: 50.00 for the nebuliz-ing gas, 50.00 for the auxiliary gas, and 20.00 for the curtain gas. Theion spray was set at 5500 V, the declustering potential was at 121 V,and the entrance potential was at 10 V. The dwell time was 150 ms,and the temperature of the heated nebulizer temperature was 400°C.The mass spectrometer was programmed to admit protonated mol-ecules [M�H]� at m/z 392.03 (I) and 552.24 (II) via the first quad-ripole filter (Q1), with collision-induced fragmentation at Q2 (colli-sion gas nitrogen, setting CAD 10.00), and monitoring the productions via Q3 at m/z 136.10 (I) and 202.20 (II). Peak area ratiosobtained from selective reaction monitoring of analytes (m/z392.03 � 128.10)/(m/z 552.24 � 202.20) were used for the construc-tion of the calibration curve, using weighed (1/x) regression of theplasma concentrations and the measured peak area ratios. Data collec-tion, peak integration, and calculations were performed using Analyst1.5 PE-Sciex software (Applied Biosystems, Foster City, CA).

Microsomal clearance was determined essentially as describedpreviously (Kuhlmann et al., 2010).

In Vivo Receptor Occupancy Measurement. Adult maleC57BL/6 mice (approximately 30 g body weight) received oral dosesof vehicle or CTEP in a range of 0.01 to 3.0 mg/kg 90 min before a tailvein injection of 0.3 mCi/kg [3H]ABP688 (specific activity: 80 Ci/mmol) formulated in 28% (w/v) N-methyl pyrrolidone in 0.9% (w/v)saline containing �2% (w/v) ethanol. After 30 min mice were sacri-ficed, plasma samples were collected as described above, and brainswere removed. From each animal, half of the brain was immediatelyfrozen on dry ice for histology and autoradiography, the remaininghalf was used for analysis of drug exposure. Parasagittal brainsections were cut at 10-m thickness on a Leica CM1950 cryostat(Leica Microsystems, Nunningen, Switzerland), collected on Super-frost plus glass slides (Menzel, Braunschweig, Germany), air-dried,exposed for 5 days on [3H]-sensitive phosphorimaging plates (GEHealthcare), and analyzed on an ImageQuant phosphorimager (GEHealthcare). Autoradiograms were analyzed using ImageQuant TLsoftware (GE Healthcare). Selected brain regions were manuallydefined based on stereotactical information (Paxinos and Franklin,2003), and radioactivity bound in these regions was quantified. Us-ing the radioactivity obtained in animals treated with vehicle and 3mg/kg of CTEP as total and nonspecific binding, IC50 values based ondrug exposure were calculated using XLfit software as described forradioligand binding.

Measurement of Physicochemical Properties. The octanol/waterdistribution coefficient was determined as described previously (Kansy etal., 1998). The plasma protein binding of drugs was determined as de-scribed previously (Banker et al., 2003; Wan and Holmen, 2009).

ResultsDiscovery of CTEP. CTEP (C19H13ON3F3Cl; molecular

weight � 391.78) (Fig. 1) was discovered in a medicinalchemistry effort at F. Hoffmann-La Roche Ltd. starting froma high-throughput screen of a small molecular weight com-pound library based on a Ca2� mobilization assay usinghuman mGlu5. The high-throughput screen delivered sev-eral hits from different chemical series including knownmGlu5 antagonists comprised in the compound library suchas MPEP, MTEP, and fenobam (Fig. 1), as well as compoundsstarting from which CTEP was discovered.

CTEP Potently Binds mGlu5 with Small Species Dif-ferences. The in vitro binding properties of CTEP were

characterized by saturation analyses of [3H]CTEP on mem-branes from HEK293 cells expressing human, mouse, and ratmGlu5. The saturation isotherms were monophasic at a con-centration range of 0.12 to 140 nM [3H]CTEP and fitted bestwith a one-site model (Fig. 2, a–c). [3H]CTEP binds to hu-man, mouse, and rat mGlu5 with a dissociation constant (Kd)of 1.7, 1.8, and 1.5 nM, respectively (Table 3). In competitionbinding experiments, CTEP fully displaced [3H]MPEP with Ki

values of 16.4 nM (human), 9.5 nM (mouse), and 12.6 nM (rat)(Fig. 2d; Table 4). [3H]CTEP is not displaced from mGlu5 by theorthosteric ligands glutamate, quisqualate, (S)-4-carboxy-3-hy-droxyphenylglycine, and (S)-3,5-dihydroxyphenylglycine (datanot shown).

CTEP Is a Negative Allosteric mGlu5 Modulator withInverse Agonist Activity. In HEK293 cells stably express-ing human mGlu5, CTEP inhibits quisqualate-induced Ca2�

mobilization with an IC50 of 11.4 nM and [3H]IP accumula-tion with an IC50 of 6.4 nM (Fig. 3b; Table 3). In the [3H]IPaccumulation assay, CTEP inhibits the constitutive activityof human mGlu5 by approximately 50% with an IC50 of 40.1nM (Fig. 4a), demonstrating inverse agonist activity as re-ported previously for MPEP and fenobam (Porter et al.,2005). It is noteworthy that orthosteric antagonists such as(S)-4-carboxyphenylglycine do not show inverse agonist ac-tivity (Porter et al., 2005).

The mode of action of CTEP was further tested by record-ing concentration-response curves for quisqualate-induced[3H]IP accumulation in the presence of various concentra-tions of CTEP. Increasing concentrations of CTEP trigger aright shift of the quisqualate concentration-response curvewith a simultaneous reduction of the maximal response am-plitude (Fig. 4b), which is indicative of an allosteric mode ofaction. This observation is in line with the lack of displace-ment of [3H]CTEP by orthosteric mGlu5 ligands.

CTEP Is Highly Selective for mGlu5. The selectivity ofCTEP with respect to other mGlu receptors was assessed formGlu1, mGlu2, and mGlu8 with Ca2� mobilization assays(Fig. 5, a, b, and g), for mGlu3 with a (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY354740)radioligand binding assay (Fig. 5c), for mGlu4 and mGlu6with a [35S]GTP�S binding assay (Fig. 5, d and e), and formGlu7 with a cAMP accumulation assay (Fig. 5f). At concen-trations up to 10 M CTEP does not show significant activity.

The selectivity profile of CTEP was extended to a total of103 targets including receptors, enzymes, and ion channelsby testing the compound in a CEREP Diversity Profile at asingle concentration of 1 M in duplicates and following uptargets showing an effect size of �20% control inhibition with

Fig. 1. Chemical structures of CTEP, MPEP, MTEP and fenobam.

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concentration-response curves (Table 2; Supplemental TableS1). Taken together, CTEP shows �1000-fold selectivity formGlu5 on all targets tested.

Anxiolytic In Vivo Activity of CTEP. CTEP was testedin two paradigms sensitive to antianxiety drugs, the SIH andthe Vogel conflict drinking tests. In the former, CTEP wassignificantly active at doses of 0.1 and 0.3 mg/kg (F3,34 � 4.9;

0.1 mg/kg, p � 0.05; 0.3 mg/kg, p � 0.001) (Fig. 6a; all dosesoral), whereas only a trend of activity was detected at a doseof 0.03 mg/kg. CTEP did not cause a significant effect on T1at all doses (data not shown). In the Vogel conflict drinkingtest, CTEP significantly increased drinking time at doses of0.3 and 1.0 mg/kg (p � 0.01), whereas it has no effect at lowerdoses (Fig. 6b; all doses oral).

Fig. 2. Saturation analysis of [3H]CTEP(a–c) and [3H]MPEP displacement bind-ing of CTEP on membranes of HEK293cells transiently transfected with human,mouse, or rat mGlu5 (d). a to c, saturationanalyses were performed in four indepen-dent experiments in triplicate. One rep-resentative experiment for each species isshown. Error bars represent S.D. d,[3H]MPEP displacement binding experi-ments were performed 12 (human,mouse) and 16 (rat) independent experi-ments in duplicate. Error bars representS.E.M.

Fig. 3. Concentration-dependent inhibi-tion of quisqualate-induced mGlu5 acti-vation by CTEP measured by IP accumu-lation (a) and Ca2� mobilization (b). a, IPaccumulation experiments were per-formed in four (human), eight (mouse),and four (rat) independent experimentsin duplicate. b, Ca2� mobilization experi-ments were performed in 16 (human), 14(mouse), and 8 (rat) independent experi-ments in duplicate. Error bars representS.E.M.

TABLE 3CTEP in vitro activity on recombinant human, mouse, and rat mGlu5 in radioligand binding and functional assaysThe affinity of CTEP for mGlu5 was determined by recording �3H�CTEP saturation isotherms (Fig. 2, a–c), and the Ki values were measured by �3H�MPEP displacementbinding on recombinant mGlu5 expressed in HEK293 cells (Fig. 2d). IP accumulation and Ca2� mobilization were tested with HEK293 cells stably (human and rat) ortransiently (mouse) transfected with plasmids encoding mGlu5 (Fig. 3). In radioligand binding experiments, data are mean S.E.M. of at least four independent experimentseach conducted in triplicate (saturation isotherms) or duplicate (displacement binding). In IP accumulation experiments, data are mean S.E.M. of four (human and rat)and eight (mouse) independent experiments each performed in duplicate. In Ca2� mobilization experiments, data are mean S.E.M. of 16 (human), 14 (mouse), and 8 (rat)independent experiments each performed in duplicate.

Species�3H�CTEP Saturation Isotherms

�3H�MPEP Competition Binding (Ki)Functional Assays (IC50)

Kd Bmax IP Accumulation Ca2� Mobilization

nM pmol/mg protein nM nM

Human 1.7 0.1 7.6 0.4 16.4 1.8 6.4 0.9 11.4 0.6Mouse 1.8 0.23 1.4 0.21 9.5 0.6 16.8 3.4 42.4 3.2Rat 1.5 0.2 7.1 0.6 12.6 1.5 8.8 1.0 6.9 0.7

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Pharmacokinetic and Physicochemical Propertiesof CTEP. The pharmacokinetic properties of CTEP in micewere studied after single and chronic administration. Aftersingle oral doses of 4.5 and 8.7 mg/kg CTEP formulated asmicrosuspension in a saline/Tween vehicle administrated toadult C57BL/6 mice was rapidly absorbed and achieved close tomaximal exposure after approximately 30 min (Fig. 7, a and b;Table 4). The half-life of CTEP (oral) was 18 h, and the B/P ratiobased on total drug concentrations in plasma and whole brainhomogenates was 2.6 (Fig. 7, a and b; Table 4). Chronic admin-istration in adult mice with a dose of 2 mg/kg p.o. every 48 h for2 months reached a minimal CTEP brain exposure of 240 ng/g(Fig. 8a). Continuous drug exposure was also achieved in new-born mice when CTEP was administrated every 48 h subcuta-neously (Fig. 8b).

For comparison, the pharmacokinetic profiles of MPEP,MTEP, and fenobam in adult C57BL/6 mice were recordedafter oral and intravenous administration (Fig. 7, c-e; Table4). When dosed orally at 30 mg/kg and formulated as micro-suspension (vehicle: 0.9% NaCl and 0.3% Tween 80), MPEP,MTEP, and fenobam were rapidly absorbed with Tmax � 0.6 h(MPEP) and 0.3 h (MTEP and fenobam). All three compoundsshowed a rapid in vivo clearance with an estimated half-lifeof 2 h (MPEP), 0.5 h (MTEP), and 0.3 h (fenobam). Thehalf-life after intravenous administration was estimated as1.9 h (MPEP), 0.2 h (MTEP), and 0.3 h (fenobam). The ratiobetween exposure in brain and plasma measured 1 h after asingle oral dose of 30 mg/kg was B/P � 0.8 (MPEP), 0.6(MTEP), and 2.7 (fenobam). The oral bioavailability of CTEPwas approximately 100% compared with approximately 6%for MPEP, 7% for fenobam, and 87% for MTEP (Table 4).

With respect to physicochemical properties, CTEP wasmoderately lipophilic with an octanol/water distribution co-efficient of logD �4 at pH 7.4. The solubility of CTEP in 50mM phosphate buffer, pH 6.5 was approximately 1 g/ml atroom temperature, and the solubility in dimethyl sulfoxidewas �100 mM. The maximum solubility in artificial cerebro-spinal fluid at pH 7.4 achieved with repeated sonication andvigorous shaking was approximately 30 g/ml at 30°C. Basedon its chemical properties, the solubility of CTEP in aqueoussolution is expected to be pH-dependent with increasing sol-ubility at lower pH. CTEP showed a high plasma proteinbinding of 99% (mouse plasma; Table 4).

In Vivo mGlu5 Brain Receptor Occupancy. ThemGlu5 receptor occupancy by CTEP was measured in brainof adult C57BL/6 mice using [3H]ABP688 (Hintermann et al.,2007), a selective mGlu5 antagonist previously used for ro-

dent in vivo binding and human positron emission tomogra-phy studies (Ametamey et al., 2007). CTEP fully displaced[3H]ABP688 in brain regions known to express mGlu5(Fig. 9), and 50% displacement was achieved with dosesproducing an average compound concentration of 77.5 ng/gmeasured in whole brain homogenate.

DiscussionPharmacological inhibitors of mGlu5 have been vital for

the current understanding of its physiological role in theCNS and enabled clinical research exploring the therapeuticpotential of mGlu5 as a drug target in the context of CNS-related disorders including anxiety, depression, pain, Parkin-son’s disease, Huntington’s disease, and fragile X syndrome(Gasparini et al., 2008; Jaeschke et al., 2008). The firstnonamino acid, selective, and noncompetitive mGlu5 antag-onists were 6-methyl-2-(phenylazo)-3-pyridinol (SIB-1757)and (E)-2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893),which inhibited human mGlu5 in IP accumulation assayswith moderate potencies of IC50 � 3.1 and 2.3 M, respec-tively (Varney et al., 1999). Structural optimization aroundthese two compounds led to the discovery of MPEP as thefirst highly potent and selective mGlu5 antagonist with apotency of IC50 of 36 nM on human mGlu5 (Gasparini et al.,1999), acting on a specific binding pocket in the transmem-brane domain of the mGlu5 receptor involving transmem-brane helices 3, 5, 6, and 7 (Pagano et al., 2000; Malherbe etal., 2003). The discovery of MPEP was a breakthrough be-cause it allowed for the first time the systemic administra-tion of an mGlu5 antagonist with good brain penetration,achieving significant in vivo efficacy in models of pain andanxiety (Kuhn et al., 2002). Further structural optimizationled to the identification of MTEP (Cosford et al., 2003) withimproved selectivity and bioavailability. The mGlu5 antago-nist fenobam was examined for its antianxiety potential inphase II clinical trials in the 1970s, although the moleculartarget was unknown at the time (Pecknold et al., 1982). Theclinical development of fenobam as an anxiolytic was discon-tinued because of side effects and erratic bioavailability (Itilet al., 1978; Friedmann et al., 1980). In 2005, mGlu5 wasidentified as the drug target of fenobam in a high-throughputscreening campaign at F. Hoffmann-La Roche Ltd., and fur-ther characterization confirmed fenobam as a potent andselective mGlu5 negative allosteric modulator and potentanxiolytic in animals (Porter et al., 2005). Even though struc-turally diverse, all three tool compounds MPEP, MTEP, and

Fig. 4. Inverse agonism (a) and noncompet-itive mode of action (b) of CTEP demon-strated on human mGlu5. a, CTEP inverseagonist property was observed by recordingits concentration-dependent inhibition ofbaseline IP formation in HEK293 cells sta-bly transfected with human mGlu5 in theabsence of an agonist. b, CTEP noncompet-itive mode of action revealed by Schildanalysis, recording concentration-responsecurves of quisqualate-induced IP formationin HEK293 cells stably transfected withhuman mGlu5 in the presence of variousconcentrations of CTEP. For both experi-ments, results were obtained in four inde-pendent experiments run in duplicate. Er-ror bars represent S.E.M.

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fenobam, act on the same binding pocket at the mGlu5 re-ceptor (Malherbe et al., 2006).

Here is described CTEP, a novel, potent, and selective orallybioavailable mGlu5 negative allosteric modulator with inverseagonist properties. In saturation analyses, [3H]CTEP binds to asingle saturable binding site on recombinant human, mouse,and rat mGlu5 receptor with high affinity. In competition bind-ing experiments with recombinant human, mouse, and rat

mGlu5, CTEP displaces [3H]MPEP with low nanomolar Ki val-ues. The full displacement of [3H]MPEP (Fig. 2d), as well as[3H]MTEP, [3H]fenobam, and [3H]ABP688 (data not shown),from recombinant mGlu5 suggests that CTEP acts on the samebinding pocket described for the three previously known mGlu5antagonists. In cellular in vitro assays, CTEP potently inhibitsIP accumulation and Ca2� mobilization triggered by quis-qualate in HEK293 cells expressing human, mouse, or rat

Fig. 5. Lack of CTEP activity on mGlu1–4and mGlu6–8. CTEP tested in concentra-tions up to 10 M does not significantlyinhibit human mGlu1 (a), human mGlu2(b), human mGlu3 (c), rat mGlu4 (d), ratmGlu6 (e), human mGlu7 (f), and humanmGlu8 (g). (�)-Ethyl (7E)-7-hydroxyimino-1,7a-dihydrocyclopropa[b]chromene-1a-carboxylate (CPCCOEt): group I mGlu an-tagonist. LY341495: mGlu2/3 antagonist.(RS)-�-methylserine-o-phosphate (MSOP):group III mGlu antagonist. At least threeindependent experiments were performedin duplicate. Error bars represent S.E.M.

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mGlu5. The lower potency of CTEP observed in functional as-says on mouse compared with human and rat mGlu5, in con-trast to the fairly consistent potency of CTEP across all threespecies in radioligand binding experiments (Table 3), was un-expected because there are no species differences in the aminoacid sequence of the mGlu5 transmembrane regions harboringthe presumed binding pocket (Supplemental Fig. S1). One pos-sible reason could be the use of transiently transfected cells for

mouse mGlu5 as opposed to stably transfected cell lines forhuman and rat mGlu5 in the functional assays. CTEP sup-pressed the constitutive activity of mGlu5 in the IP accumula-tion assay (Fig. 4a), revealing inverse agonist properties similarto MPEP and fenobam (Porter et al., 2005). The allosteric modeof action of CTEP was demonstrated by the rightward shift ofagonist concentration-response curves and a simultaneous re-duction of the maximal agonist response in the Schild plot (Fig.4b). These observations are in line with CTEP fully displacingother allosteric mGlu5 ligands such as [3H]MPEP, [3H]MTEP,[3H]fenobam, and [3H]ABP688 and with the lack of [3H]CTEPdisplacement by orthosteric ligands (data not shown). Screen-ing a total of 103 molecular targets, including all knownmetabotropic glutamate receptors, revealed �1000-fold selec-tivity of CTEP for mGlu5.

In vivo, CTEP showed robust pharmacological activity intwo models sensitive to antianxiety drugs, the SIH procedurein mice and the Vogel conflict drinking method in rats, witha minimal effective oral dose of 0.1 and 0.3 mg/kg, respec-tively (Fig. 6). Compared with fenobam and MPEP, CTEPshows a 30- to 100-fold higher in vivo potency in these pro-cedures (Ballard et al., 2005; Porter et al., 2005). The in vivoexperiments reported here did not reveal any unspecific ef-

Fig. 6. Activity of CTEP in two behavioral models sensitive to antianxietydrugs. CTEP was dose-dependently active in the SIH procedure in mice(a) and the Vogel conflict drinking test in rats (b). Data are mean S.E.M. based on 10 to 12 animals per group. �, p � 0.05; ��, p � 0.01; ���,p � 0.001 versus vehicle.

Fig. 7. Pharmacokinetic profiles of CTEP,MPEP, MTEP, and fenobam in C57BL/6mice. a and b, the pharmacokinetic profileof CTEP in plasma (a) and brain (b) ofadult C57BL/6 mice after a single oraladministration of 4.5 and 8.7 mg/kg. c toe, the pharmacokinetic profiles of MPEP(c), MTEP (d), and fenobam (e) in plasmaafter a single dose of 10 mg/kg i.v. and 30mg/kg p.o. f, side-by-side comparison ofthe pharmacokinetic profiles of CTEP,MPEP, MTEP, and fenobam adminis-trated orally to adult mice. The recordedplasma profiles represent composite pro-files with combined data of typically fiveto seven animals per compound, dose, androute (n � 2 analyzed samples for eachdata point).

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fects of CTEP such as sedation or general changes in overallhealth, body temperature, and body weight in a dose range ofup to 2 mg/kg. Because of the large differences in the sensi-tivity of species and test procedures, e.g., for effects of mGlu5inhibitors on learning and memory (Simonyi et al., 2005), thepossible target-mediated or target-unrelated unspecific ef-fects of CTEP cannot be generally ruled out at this point.Pharmacokinetic studies of CTEP in C57BL/6 mice revealeda high oral bioavailability of approximately 100% and goodbrain penetration with an average B/P ratio of 2.6 measuredas total drug concentrations in plasma and whole brain ho-mogenate, respectively. In this context it is important to notethat the reported plasma and brain tissue drug concentra-tions are not identical to the free fraction of the compoundavailable for binding to the target and do not reflect thesubcellular distribution of the drug known to influence itspharmacology (Jong et al., 2009). These results are in linewith CTEP in vivo binding data revealing a 50% displace-ment of the tracer [3H]ABP688 from cortex, hippocampus,

and striatum with a dose producing an average drug concen-tration of 77.5 ng/g in whole brain homogenates and achiev-ing full displacement by a single oral dose of 3 mg/kg (Fig. 9).

One distinctive feature of CTEP is the long half-life of 18 hcompared with half-lives of �2 h for MPEP, MTEP, andfenobam (Table 4). On this basis, continuous drug exposurefor chronic treatment can be achieved with one dose of CTEPper 48 h in adult and newborn mice (Fig. 8), whereas thereference compounds MPEP, MTEP, and fenobam would re-quire multiple doses per day. Beyond logistical and cost con-siderations, long intervals between drug administrations aregenerally preferred for chronic treatment because the fre-quent manipulation of animals can affect behavioral andother parameters, alter the basic phenotype (e.g., of trans-genic mouse models), and even influence overall well-beingand survival. For example, vehicle or sham injections canimpair the performance of mice in the elevated plus maze test(Lapin 1995). Frequent handling of animals can resemble orcontribute to enriched environment housing conditions that

Fig. 8. CTEP exposure achieved with chronic dosing in mice. CTEP brain exposure measured in adult (a) and newborn (b) mice after multiple drugadministration. CTEP was administered once per 48 h at 0.7 and 2 mg/kg, and brain samples were collected 48 h after dosing. Adult male C57BL/6mice received CTEP orally, and newborn mice were 2 days old at first dosing and received subcutaneous CTEP injections. Data represent mean of twosamples analyzed per time point and experiment. Because exposure was measured 48 h after each administration, data represent the trough (i.e.,minimal) exposure levels during the dosing period.

TABLE 4Pharmacokinetic properties of CTEP, MPEP, MTEP, and fenobam in adult mice and in human and mouse liver microsomesAll data except microsomal clearance are derived from composite pharmacokinetic profiles, with profiles for each compound and route constructed with data from multipleanimals (plasma data adult mice, two consecutive plasma samples per animal; brain data adult mice, one brain sample per animal; Microsomal clearance is mean of n �2 S.D.

AllostericModulator Dose Cmax Tmax T1/2 Clearance Vss

OralBioavailability

B/PRatio

ProteinBinding

Clearance inMicrosomes

Human Mouse

mg/kg ng/ml h ml/min/kg l/kg % % l/min/mg proteinat 1 M

CTEP 3.0 2.8 4.5 2.1Oral 4.5 (8.7)a 716 7.6 18b (55)a �100c 2.6 99Intravenous 1.9 771 0.08 34 2.1 5.8 2.6 99

MPEP 276.5 17.7 424.0 86.9Oral 37.7 912 0.6 N.A. �6d 0.8e 99Intravenous 18.1 3930 0.1 1.9 206 4.8 0.8e 99

MTEP 60.5 2.1 401.5 33.2Oral 34.9 7435 0.3 0.5 87 0.6e 85Intravenous 11.1 6750 0.1 0.2 82 0.8 0.6e 85

Fenobam 46.0 4.2 442.0 5.7Oral 31.9 796 0.3 0.7 7f 2.7e 73Intravenous 10.3 8410 0.2 0.4 33 0.7 2.7e 73

N.A., not available.a The longer T1/2 obtained with a higher dose indicates a nonlinear clearance at higher doses.b T1/2 calculated in a time interval 48 to 96 h postdose; for discussion and further calculations, the CTEP half-life of 18 h obtained with an oral dose of 4.5 mg/kg is used

because this dose is closer to the doses used in behavioral assays and for chronic drug treatment.c In view of dose-dependent nonlinear clearance observed with high doses, the oral bioavailability values represent a rough estimate.d Based on truncated area under the curve (0–7 h).e Ratio calculated from values recorded 1 h after oral dosing of a dose of 30 mg/kg for each compound.f Estimate.

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have been reported to profoundly influence an animal’s phe-notype and behavior, e.g., the increase of synaptic spinedensity and the elevation of brain-derived neurotrophic fac-tor expression and rate of neurogenesis (Ickes et al., 2000;Nithianantharajah and Hannan, 2006). Furthermore, en-riched environment has been reported to reverse multiplephenotypes in a mouse model of fragile X syndrome (Restivoet al., 2005), ameliorate impaired learning and memory per-formance in a mouse model of Down syndrome (Martínez-Cue et al., 2002), and delay the loss of dopaminergic neuronsin models of Parkinson’s disease (Faherty et al., 2005). Onthis background, the use of drugs with a long half-life such asCTEP allowing the least dosing frequency for chronic treat-ment offers advantages especially when dealing with fragileor very young animals and with phenotypes or parameterssensitive to enriched environment.

Taken together, CTEP represents a novel, potent, and se-lective orally bioavailable mGlu5-negative allosteric modula-tor with inverse agonist activity, and it represents the firstmGlu5 inhibitor with a long half-life. By enabling long dura-tion treatments through a wide age range, CTEP is expectedto make an important contribution to the efforts of exploring thefull therapeutic potential of mGlu5 inhibitors for indications

such as Parkinson’s disease and depression and for neurodevel-opmental disorders including fragile X syndrome and autism.

Acknowledgments

We thank Sean Durkin, Brigitte Algeyer, Antonio Ricci, DanielRueher, Celine Sutter, Patricia Glantzlin, Christelle Rapp-Mary,Daniela Doppler, Hugues Isel, Thomas Thelly, Roland Degen,Karina Muller, Mathias Muller, and Volkmar Starke for excellenttechnical assistance and Marius Honer, Sylvie Chabos, Daniele Bu-chy, Paricher Malherbe, Anne Marcuz, Lucinda Steward, GabriellePy, Christoph Ullmer, Anja Osterwald, Monique Dellenbach, andJennifer Beck for contributions to the selectivity screen.

Authorship Contributions

Participated in research design: Lindemann, Jaeschke, Honer, andPorter.

Conducted experiments: Lindemann, Jaeschke, Michalon, Honer,Porter, Buttelmann, Flament, Diener, Fischer, Gatti, Prinssen, andHoffmann.

Contributed new reagents or analytic tools: Lindemann, Jaeschke,Vieira, Hartung, and Kolczewski.

Performed data analysis: Lindemann, Jaeschke, Michalon, Honer,Porter, Hartung, Flament, Diener, Fischer, Gatti, Prinssen, Parrott,and Hoffmann.

Wrote or contributed to the writing of the manuscript: Lindemann,Jaeschke, Michalon, Spooren, and Wettstein.

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Address correspondence to: Dr. Lothar Lindemann, F. Hoffmann-La RocheLtd. Pharmaceuticals Division, Discovery Neuroscience, Grenzacherstrasse124, 4070 Basel, Switzerland. E-mail: [email protected]

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