European Journal of Pharmacology 627 (2010) 106–114
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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r
Neuropharmacology and Analgesia
In vitro and in vivo comparison of two non-peptide tachykinin NK3 receptor antagonists: Improvements in efficacy achieved through enhanced brain penetration or altered pharmacological characteristics Lee A. Dawson 1, Christopher J. Langmead, Adeshola Dada, Jeannette M. Watson, Zining Wu, Raúl de la Flor, Gareth A. Jones, Jane E. Cluderay, Eric Southam, Graham S. Murkitt, Mark D. Hill, Declan N.C. Jones, Ceri H. Davies, Jim J. Hagan, Paul W. Smith ⁎ Neurosciences Centre of Excellence for Drug Discovery, GlaxoSmithKline, New Frontiers Science Park, Harlow, Essex, UK
a r t i c l e
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Article history: Received 11 June 2009 Received in revised form 9 October 2009 Accepted 26 October 2009 Available online 30 October 2009 Keywords: Microdialysis Dopaminergic neurotransmission Nucleus accumbens Prefrontal cortex Guinea pig Tachykinin Pharmacokinetic Non-surmountable
a b s t r a c t Clinical evaluation of tachykinin NK3 receptor antagonists has provided support for the therapeutic utility of this target in schizophrenia. However, these studies have not been entirely conclusive, possibly because of the pharmacokinetic limitations of these molecules. In the search for tachykinin NK3 receptor antagonists with improved properties, we have discovered GSK172981 and GSK256471. Both compounds demonstrated high affinity for recombinant human (pKi values 7.7 and 8.9, respectively) and native guinea pig (pKi values 7.8 and 8.4, respectively) tachykinin NK3 receptors. In vitro functional evaluations revealed GSK172981 to be a competitive antagonist (pA2 = 7.2) at cloned human tachykinin NK3 receptor whereas GSK256471 diminished the neurokinin B-induced Emax response, indicative of non-surmountable antagonist pharmacology (pA2 = 9.2). GSK172981 also exhibited a competitive profile in antagonizing neurokinin B-stimulated neuronal activity recorded from the guinea pig medial habenula slices (apparent pKB = 8.1), whilst GSK256471 abolished the agonist-induced response. Central nervous system penetration by GSK172981 and GSK256471 was indicated by dose-dependent ex vivo tachykinin NK3 receptor occupancy in medial prefrontal cortex (ED50 values of 0.8 and 0.9 mg/kg, i.p., respectively) and the dose-dependent attenuation of agonist-induced “wet dog shake” behaviours in guinea pigs. Finally, in vivo microdialysis studies demonstrated that acute GSK172981 (30 mg/kg, i.p.) and GSK256471 (1 mg/kg, i.p.) attenuated haloperidol-induced increases in extracellular dopamine in the guinea pig nucleus accumbens. Taken together, these in vitro and in vivo characterisations of the tachykinin NK3 receptor antagonists GSK172981 and GSK256471 support their potential utility in the treatment of schizophrenia. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The NK3 receptor is one of the tachykinin peptide neurotransmitter receptor family for which neurokinin B is the endogenous ligand. Tachykinin NK3 receptors are expressed in neurons of the peripheral and central nervous systems (Mileusnic et al., 1999; Langlois et al., 2001; Rigby et al., 2005). In the mammalian brain low level expression is seen in frontal, parietal and cingulate cortices, amygdala, hippocampus, substantia nigra, ventral tegmental area, raphe nucleus, locus coeruleus and septal and basal nuclei (Mussap et al., 1993; Tooney et al., 2000; Langlois et al., 2001). Consistent with this distribution
⁎ Corresponding author. Neurosciences CEDD, GlaxoSmithKline, New Frontiers Science Park (North), Harlow, Essex, CM19 5AW, UK. Tel.: +44 1279 622000. E-mail addresses:
[email protected] (L.A. Dawson),
[email protected] (P.W. Smith). 1 Current address: Eisai Ltd., Eisai European Knowledge Centre, Mosquito Way, Hatfield, Hertfordshire, AL10 9SN, UK. 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.10.054
pattern, agonist-induced tachykinin NK3 receptor activation stimulates noradrenergic (Jung et al., 1996) and dopaminergic cell firing (Nalivaiko et al., 1997) in the locus coeruleus and ventral tegmental area, respectively and increases dopamine levels in the nucleus accumbens and striatum (Alonso et al., 1996; Marco et al., 1998; Preston et al., 2000) and noradrenaline in the medial prefrontal cortex (Jung et al., 1996; Bert et al., 2002). Conversely, systemic administration of the tachykinin NK3 receptor antagonist talnetant (SB223412) produces an elevation in prefrontal cortex dopamine and hippocampal noradrenaline levels (Dawson et al., 2008). Tachykinin NK3 receptor antagonists also attenuate haloperidol-induced A9/A10 dopaminergic cell firing (Gueudet et al., 1999) and haloperidolinduced dopamine efflux in the nucleus accumbens, but without affecting basal output per se (Sarau et al., 1997; Gueudet et al., 1999; Dawson et al., 2008). Thus tachykinin NK3 receptor antagonists have the potential to reverse both hypofunction within the prefrontal cortex and hyperfunction of the mesolimbic dopamine system, mechanistic effects that would be predicted to bring about beneficial
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effects in the treatment of the cognitive and positive symptoms of schizophrenia, respectively. Recently this hypothesis has been partially supported by the data from a tachykinin NK3 receptor knockout mouse. Nordquist et al. (2008a) evaluated these mice in delayed matching to position, spontaneous alternation, water maze and active avoidance paradigms and demonstrated an improved operant performance (but not working memory per se) and a more rapid response latency. A follow on study (Nordquist et al., 2008b) demonstrated no overt change in behavioural responsiveness to amphetamine and some quite subtle changes in markers of dopamine function in these mice. However, and in contrast, an alternatively derived knockout mouse (Siuciak et al., 2007) actually showed deficits in both acquisition and retention performance in some of these cognition paradigms. The reason for this difference is unclear at present. However the hypothesis has recently gained credence with the appearance of preliminary clinical investigations of tachykinin NK3 receptor antagonists in schizophrenia patients. SR142801 (osanetant) showed efficacy in four primary outcome measures (Positive and Negative Scale for Schizophrenia (PANSS) total, Brief Psychiatric Rating Scale (BPRS), BPRS positive symptom subscale (delusions, hallucinations, conceptual disorganization and bizarre behaviour) and the Clinical Global Impression Scale (CGI-S) in a 6 week multi-centre, double-blind, randomised clinical study in hospitalised schizophrenic and schizoaffective patients (Meltzer et al., 2004). Furthermore, the improvement in these patients was not significantly different from that achieved with the antipsychotic haloperidol (Meltzer et al., 2004). However, it should be noted that, although not published in peer reviewed format, a further study failed to reproduce these initial findings (Meltzer and Prus, 2006). Subsequent studies with talnetant also showed some efficacy against the positive and cognitive domains in schizophrenic patients (Evangelista, 2005; Meltzer and Prus, 2006). Furthermore, Liem-Moolenaar et al. (2008) showed that talnetant produced a similar effect to haloperidol (3 mg) in decreasing α-power spectra (as measured using electroencephalography (EEG)) and improved adaptive tracking and reduced calmness on the VAS Bond and Lader, whilst haloperidol impaired and reduced alertness and mood, respectively. It is obviously difficult to draw too many firm conclusions from these limited data but they do seem to demonstrate central nervous system (CNS) penetration without D2-like side effects. One could further speculate that the observed effects may be dopaminergic in nature (i.e. increase in attention (enhanced cortical dopamine seen preclinically; Dawson et al., 2008) and reduced calmness). But these data do seem to largely support the clinical observations to date. However, the lack of a definitive clinical signal may be a consequence of the less than ideal pharmacokinetic characteristics of both talnetant and osanetant (Meltzer and Prus, 2006). We now report the in vitro and in vivo characterisation of 3amino-N-[(S)-cyclopropyl (phenyl)methyl]-2-(3-fluorophenyl)4-quinolinecarboxamide (GSK172981) and N-[(S)-cyclopropyl (3-fluorophenyl)methyl]-3-{[methyl(methylsulfonyl)amino]methyl}2-phenyl-4-quinolinecarboxamide (GSK256471; Smith et al., 2009; Fig. 1). The preclinical profiles of both molecules support the hypothesis that tachykinin NK3 receptor antagonism may offer therapeutic utility in the treatment of psychiatric disorders and may provide differing means of addressing the reported shortcomings of current clinical candidates (Spooren et al., 2005; Meltzer and Prus, 2006).
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Fig. 1. Diagramatic representations of the structures of GSK172981 and GSK256471.
phenyl-4-quinolinecarboxamide (GSK256471) were synthesised by Medicinal Chemistry, Psychiatry Centre of Excellence in Drug Discovery, GlaxoSmithKline (Harlow). 2.2. Animals Male Dunkin Hartley guinea pigs (David Hall or B&K Universal, UK) and male Sprague Dawley rats (Charles River, UK) were housed four per cage in a temperature (18 ± 2 °C) and humidity (55 ± 5%) controlled environment on a 12 h light/dark cycle with lights on at 06.00. Food and water were available ad libitum. All experiments were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986 and with GlaxoSmithKline ethical standards. 2.3. In vitro evaluation
2.1. Materials
2.3.1. Tachykinin NK1/NK2/NK3 receptor binding Binding affinities of GSK172981 and GSK256471 for the human (h) NK1, NK2 and NK3 receptors were determined using in vitro scintillation proximity assays (SPA) by measuring the displacement of: [125I]Tyr8-Substance P from Chinese Hamster Ovary (CHO) cell membranes stably expressing the human NK1 receptor; [125I]neurokinin A from HEK293 cell membranes stably expressing the human NK2 receptor; MePhe7[125I]His-neurokinin B from human embryonic kidney (HEK) 293 cell membranes stably expressing the human NK3 receptor. Briefly, polystyrene Leadseeker WGA-SPA beads (Amersham Biosciences) were mixed with plasma membrane prepared from HEK293 or CHO cells expressing cloned human tachykinin receptors in a bead/membrane ratio of 20:1 (w/w) in assay buffer (75 mM Tris pH 7.8, 75 mM NaCl, 4 mM MnCl2, 1 mM EDTA, 0.05% Chaps, 1 mM PMSF). The mixture was placed on ice for 20 min to allow the formation of the bead/membrane complex before bovine serum albumin was added to a final concentration of 1%. After a further 20 min incubation on ice, the bead/membrane complex was washed twice and suspended in assay buffer. 125I-labelled ligands were then added and 10 μl of the resulting mixture was then dispensed into wells of a low volume Greiner 384-well plate which contained 100 nl of test compound dissolved in 100% DMSO. The plates were sealed, briefly centrifuged (5 min, 1200 g), incubated for 2–3 h at room temperature with shaking, again centrifuged (2 min, 1200 g), and then analysed using a Viewlux imager (PerkinElmer) for 5 min with a 618-nm filter. Data was analyzed with non-linear curve fitting using the ActivityBase 5.0 software (ID Business Solution Limited). pIC50 values were calculated based on the fitted inhibition curves. pKi values (−log of the inhibition constant) were then calculated from the IC50 values as described by Cheng and Prusoff (1973) using Kd values for the radioligands determined in separate experiments.
3-amino-N-[(S)-cyclopropyl(phenyl)methyl]-2-(3-fluorophenyl)4-quinoline carboxamide (GSK172981) and N-[(S)-cyclopropyl(3fluorophenyl)methyl]-3-{[methyl(methylsulfonyl)amino]methyl}-2-
2.3.2. In vitro binding to guinea pig native NK3 receptors Guinea pig cerebral cortices were dissected and homogenised (Polytron, 15 s, setting 5) in 30 volumes (w/v; based on wet weight of
2. Material and methods
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tissue) of 50 mM Tris (pH 7.4 at 37 °C) and membranes prepared as above. Cerebral cortex membranes (100–200 μg protein/well) were incubated in Tris HCl buffer (50 mM, pH 7.4 at 37 °C) containing MgCl2 (10 mM) and BSA (0.1%) with the tachykinin NK3 receptor antagonist radioligands [3H]SB-222200 (for GSK172981) or [3H] SR142801 (for GSK256471) included at a concentration of 1 nM for inhibition binding and eight concentrations within the range 0.1 to 100 nM for saturation analyses in the presence or absence of the test compound for 60 min at 37 °C. For both saturation and inhibition binding analyses, non-specific binding was determined using 1 μM SB787976 (a selective, non-peptide tachykinin NK3 receptor antagonist; Smith et al., 2009). The reaction was terminated by rapid filtration through Whatman GF/B grade filters (pre-soaked with 0.3% polyethyleneimine) followed by 5 × 1 ml ice-cold buffer washes and bound radioactivity was determined by liquid scintillation counting. Data analysis was performed as for the recombinant binding studies, except that GraphPad Prism (GraphPad Software Inc.) was used for the curve fitting. Data are expressed as the mean ± S.E.M. of at least 3 separate experiments each performed using duplicate determinations.
oxygenated in the latter aCSF at a rate of 2–4 ml/min and maintained at 33 °C. Action potential recordings were collected and analysed using pClamp 9.0 software (Axon Instruments Inc.) or Spike 2 (Cambridge Electronic Design, UK). The effect of senktide was evaluated by measuring the mean action potential discharge frequency during the final 2 min of exposure to each concentration of drug, when a steadystate effect had been reached. In the case of neurokinin B, no steady state was reached, so the 60 s period of maximal activity was used to assess drug effects. Neuronal firing frequency in the presence of senktide was expressed as a percentage of baseline (pre-drug). Neurokinin B effects were normalised to the baseline (pre-drug) response to 300 nM neurokinin B. Concentration–response curves were fitted and IC50 values calculated using Origin 5.0 (Microcal) or Prism 4 (Graphpad). Estimations of affinity (pKB) were calculated using the global fitting of the Schild equation (as for the IP assay and with the Schild slope constrained to 1) for GSK172981 or according to an operational model of non-competitive antagonism (Kenakin, 2004) for GSK256471. 2.4. In vivo evaluations
2.3.3. Inositol phosphate accumulation in U2OS cells transiently expressing the human NK3 receptor Human osteosarcoma (U2OS) cells previously transduced with the human tachykinin NK3 receptor (utilising a recombinant Baculovirus under the control of a mammalian promoter generated using vectors and methods described in Ames et al., 2004) were incubated for 16 h with inositol-free DMEM in the presence of 1 μCi [3H]myo-inositol per well (Amersham U.K). The growth medium was aspirated, and the cells were washed with 2 × 200 μl inositol-free DMEM/3% BSA and then pre-incubated (30 min, 37 °C) in the absence or presence of antagonist before the addition of neurokinin B (0.1 nM–10 μM; 30 min, 37 °C, in the presence of 5 mM LiCl) in a final assay volume of 200 μl. In experiments designed to investigate the reversibility of antagonism, cells were briefly washed three times prior to the addition of neurokinin B. The assay was terminated and inositol phosphates (IPx) extracted by the addition of 0.1 M formic acid (200 μl) and 20 μl aliquots were transferred to Picoplates (PerkinElmer Life Sciences) containing 80 μl of RNA-Ysi SPA beads (Amersham, U.K.) previously diluted 1:8 (v/v) with double de-ionised water. The Picoplates were shaken for 60 min and then centrifuged (10 min, 1000 g) and counted using a scintillation counter. [3H]IPx accumulation, expressed as a percentage of the maximal neurokinin B response, was plotted versus concentration of test compound and was fitted globally to the Schild equation (Motulsky and Christopoulos, 2003) using GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA) to determine a pA2 value and Schild slope for GSK172981. For experiments with GSK256471 where the maximal response was diminished, the value of the maximal asymptote was allowed to vary to allow an empirical estimation of antagonist affinity (pA2). Data are expressed as mean ± S.E.M. of at least three separate experiments. 2.3.4. In vitro electrophysiology Guinea pigs were decapitated and their brains rapidly removed and immersed in an ice-cold sucrose-containing artificial cerebrospinal fluid (aCSF) composed of (mM): sucrose (189), glucose (10), NaHCO3 (26), KCl (2.5), MgCl2 (5), CaCl2 (0.1), NaH2PO4 (1.2) and bubbled continuously with an O2/CO2 mixture (95/5%) to maintain a pH of 7.4. Coronal slices (400 μm) containing the medial habenula were cut using a Vibroslice (Camden Instruments Inc) and then immersed in aCSF containing (mM): NaCl (120), KCl (2.5), CaCl2 (1.2), MgCl2 (1.5), NaHCO3 (25), NaHPO4 (2.5), glucose (10), again bubbled with 95/5% O2/CO2, for at least 30 min prior to initiation of recording. A single slice was then transferred to a recording chamber (submerged configuration) which was continuously perfused with
2.4.1. Autoradiographical determination of cortical ex vivo receptor occupancy Guinea pigs were administered vehicle (1% methylcellulose, 2 ml/kg), GSK172981 (1–30 mg/kg) or GSK256471 (1–100 mg/kg; n =4 per group) via intraperitoneal (i.p.) injection. One hour post dose, animals were sacrificed and brains were rapidly removed and frozen in isopentane maintained at approximately −40 °C. Hindbrain and trunk blood samples were collected and analysed for drug concentration (see below). Subsequently, 14μm coronal sections, from between 15.4 and 15.8 mm anterior to the frontal zero plane, as defined by Rapisarda and Bacchelli (1977), were cut using a Leica CM3050 cryostat and thaw-mounted onto Superfrost Plus microscope slides (BDH) then stored at −80 °C until use. Slides were thawed and rapidly dried under a stream of cold air then subjected to a 10 min incubation at room temperature with 10 nM [3H]senktide (Perkin Elmer; 51.9 Ci/mmol) in 50 mM Tris–HCl, pH 7.5, containing 3 mM MnCl2, 0.02% (w/v) BSA (Roche), 40 μg/ml bacitracin (Sigma), 2 μg/ml chymostatin (Sigma) and 4 μg/ml leupeptin. Adjacent sections were incubated in the same solution with the addition of 10 μM SR-142801 to define non-specific binding. Slides were then subjected to a series of four short (1 min) washes in icecold 50 mM Tris–HCl, pH 7.5, followed by two rinses in ice-cold deionised water and were then rapidly dried under a stream of cold air. Bound radioligand (expressed as counts per min per square mm) was quantified by direct beta-particle imaging (Beta Imager, BioSpace) for 12 h. Specific binding was determined in medial prefrontal cortex as the mean of bilateral non-specific binding measurements from 2 sections per animal (i.e., four determinations per animal) subtracted from the mean of total binding bilateral measurements from 4 sections per animal (i.e., eight determinations per animal). Percent occupancy values were calculated using the following formula: (vehicle group mean specific binding − individual animal specific binding) ÷ vehicle group mean specific binding) × 100. Curve fitting was carried out using one-site binding non-linear regression analysis in Prism 4 (GraphPad Software Inc.). 2.4.2. Pharmacokinetic evaluation in guinea pigs and rats Rats or guinea pigs were dosed orally (p.o.; 3 mg/kg), intravenously (i.v.; 1 mg/kg) or intraperitoneally (i.p.; 10 mg/kg; guinea pigs only) with GSK172981 or GSK256471. Fresh blood and brain samples were extracted using MeCN and protein precipitation by addition of “precipitation buffer” (95:5 MeCN: EtOH containing 0.1% formic acid and an internal standard) followed by centrifugation at 1600 g for 15 min. The resulting supernatant (100 µl) was removed and analysed
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for GSK172981 or GSK256471 using liquid chromatography coupled to mass spectrometry (LC-MS-MS). Chromatographic separations were performed using gradient HPLC on Acquity UPLC™ BEH C18 columns (50 × 2.1 mm) and Waters Acquity UPLC (Waters, Mildford, USA). Separation was achieved via a gradient (mobile phase A = 5% MeCN/95% water/0.1% formic acid, mobile phase B = 95% MeCN/5% water/0.1% formic acid) transition from 85%: 15% to 5%: 95% over 1.1 min at a flow rate of 0.5 ml/min. GSK172981/GSK256471 were detected using Waters Micromass Quattro Premier VAA301 mass spectrometer (Waters, Mildford, USA) and quantified using Masslynx v 4.0 software for the PC. 2.4.3. Guinea pig “wet dog shake” behaviour Guinea pigs were anaesthetised with a 3% isoflurane and 0.5 l/min oxygen mixture. A unilateral intracerebroventricular (i.c.v.) cannula (0.25 μm diameter; co-ordinates AP = + 9.8 mm; L = ± 2.3 mm; DV = − 4.0 mm from intra-aural line and skull surface) was directed at the lateral ventricle and fixed in position using three tether screws and cyanoacrylate adhesive. Animals were allowed seven days postoperative care prior to experimentation. Before testing, guinea pigs were handled daily and habituated to clear plastic observation cages on at least five occasions. On test days, guinea pigs were administered senktide (0.3 μg, i.c.v.; Bachem, UK) and 180 s later wet dog shake behaviour was scored for 30 min by trained observers who were blind to treatment group. Senktide was dissolved in 20 mM acetic acid (0.1%)/PBS solution and administered using an injection needle extending 1 mm beyond the tip of the i.c.v. cannula. Senktide or vehicle solution was injected in a volume of 5 μl. GSK172981 (3, 10 or 30 mg/kg, i.p.; n = 9–10), GSK256471 (3, 10 or 30 mg/kg, i.p.; n = 9–10) or vehicle (1% methyl cellulose in water) were administered 60 min prior to senktide administration. Data are expressed as the mean number of episodes of wet dog shake behaviours ± S.E.M. Data were analysed by one-way ANOVA followed by post hoc Dunnet's t-test. 2.4.4. Microdialysis Guinea pigs were anaesthetised by gaseous administration of isofluorane. Microdialysis guide cannulae (CMA 11, CMA, UK) were implanted for sampling from the Nucleus accumbens. Implantation coordinates were measured from bregma (AP and ML) and the surface of the dura (DV) and were AP +4.0 mm, ML −1.5 mm and DV −6.0 mm (Rapisarda and Bacchelli, 1977). Guide cannulae were secured with dental cement and tether anchor screw (Instech, Presearch, Hitchin, UK). Animals were allowed seven days post-operative care prior to experimentation. Microdialysis was performed according to the methods described by Hughes and Dawson (2004). In brief, microdialysis probes (2 mm cuprophane membrane, CMA11 14/02, CMA, UK) were implanted and perfused with aCSF containing (mM) NaCl (145), KCl (2.7), MgCl2 (1.0), CaCl2 (1.2), Na2HPO4 (2.0) at 1 µl/min. After 2 h equilibration, four basal microdialysis samples were collected. Guinea pigs implanted with nucleus accumbens probes were initially dosed (1 ml/kg, i.p.) with a pre-treatment of vehicle (1% methylcellulose), GSK256471 (1–30 mg/kg) or GSK172981 (10 or 30 mg/kg) followed 30 min later with either haloperidol (0.5 mg/kg, i.p.) or vehicle. A 30 min sampling regime was used throughout and all microdialysate samples were collected and analysed for dopamine, norepinephrine and 5-HT content. At the end of the experiment, probes were removed and animals returned to their home cages. Animals were re-used in a randomised cross-over design with 7 days between each use and on a maximum of 4 occasions (Hughes and Dawson, 2004). After the final microdialysis experiment, animals were sacrificed and their brains were removed and stored in formalin before verification of probe placement. Brains were sectioned (50 µm) using a cooled cryostat and sections stained with cresyl violet to visualise sites of probe implantation. Data from animals with incorrect probe placement were discarded from the analysis.
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Chromatographic separations were performed using a Capcell PAK, strong cation exchange column (5 µm UG80, 1.5 × 150 mm; Shiseido, Tokyo, Japan). The mobile phase, consisted of (mM): NaCl (5) Na2HPO4 (13), NaH2PO4 (87), EDTA.Na2 (0.1), in 20% methanol buffered to pH 6, was delivered via a Jasco PU-980 HPLC pump (Jasco, Tokyo, Japan) at a flow rate of 0.2 ml/min at a temperature of 40 °C. Dopamine, noradrenaline and 5-hydoxytryptamine (5-HT) were detected via an electrochemical amperometric detector (Decade, Antec-Leyden, Leyden, Netherlands) fitted with a 3 mm glassy carbon electrode set at + 500 mV versus Ag/AgCl reference. The analogue data output was smoothed at 40 Hz (LINK, Antec-Leyden, Leyden, Netherlands) before collection. Samples (10 µl) were injected via a cooled (4 °C) Gilson model 234 autosampler (Gilson, Villiers-le-Bel, France) fitted with a six port rotary valve (Model 7125, Rheodyne, Berkeley, USA) with a 20 µl injection loop. All data were acquired using the Millenium32 software (Waters, Mildford, USA). Mean concentration of neurotransmitter in the first four baseline samples was calculated and this value denoted as 100%. Values for all samples were expressed as a percentage of this mean pre-injection control value. These transformed data were analysed by 2-way ANOVA with repeated measures followed by post-hoc Fishers test where appropriate. Post-drug administration total DA efflux was calculated as area under the curve (AUC) using a trapezoidal calculation. These transformed data were analysed by one-way ANOVA with repeated measures followed by Fisher's least squares differences test. 3. Results 3.1. In vitro binding affinity for the cloned human tackykinin NK1, receptors and guinea pig native tachykinin NK3 receptors
2, 3
Agonist radioligand competition binding assays demonstrated that both GSK172981 and GSK256471 displayed high affinity for the human tachykinin NK3 receptors with pKi values of 7.7 ± 0.1 and 8.9 ± 0.1, respectively. Compared with its affinity at the tachykinin NK3 receptor, GSK172981 showed markedly lower affinity for both tachykinin NK1 (pKi = 5.3 ± 0.1) and tachykinin NK2 (pKi = 5.9 ± 0.1) receptors whilst the affinity of GSK256471 was similarly low for tachykinin NK1 receptors (pKi 5.2 ± 0.1) but somewhat higher (pKi 7.3 ± 0.1) for tachykinin NK2 receptors. Both molecules showed moderate to high affinity (GSK172981, pKi = 7.8 ± 0.1; GSK256471, pKi = 8.4 ± 0.1) for native guinea pig cortex tachykinin NK3 receptors. 3.2. Selectivity screening of GSK172981 and GSK256471 Both GSK172981 and GSK256471 were cross-screened in external (Cerep, France; data are held on file at GSK) and in-house selectivity assays and showed at least 100-fold selectivity against up to 300 targets comprising enzymes, ion channels and 7 transmembrane receptors. These included: dopamine (D1, D2, D3, and D4) receptors, serotonin (5HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2C, 5-HT3, 5HT4, 5-HT6, and 5-HT7) receptors, noradrenergic receptors (α1 and α2) histamine (H1 and H2) receptors, muscarinic (M1 and M2) receptors, GABAA, GABAB receptors, opioid receptors and ionotropic glutamate receptors. 3.3. In vitro functional activity at human recombinant NK3 receptors as measured using IPx accumulation The functional activity of GSK172981 and GSK256471 at the human tachykinin NK3 receptor was assessed by its ability to affect neurokinin B-stimulated IPx accumulation (Brandish et al., 2003) in U2OS cells expressing the hNK3 receptor. As illustrated in Fig. 2A, GSK172981 produced a concentration-dependent, parallel rightward
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Fig. 2. The effects of (A) GSK172981 (100 nM–10 μM) and (B) GSK256471 (10 nM– 1 μM) on the neurokinin B-stimulated increase in inositol phopshate accumulation in U20S cells transiently expressing the human tachykinin NK3 receptor. Data points represent the mean ± S.E.M. from at least 3 separate experiments and are shown as the % of the fitted maximal response to neurokinin B.
shift in the neurokinin B-concentration response curve with a pA2 value of 7.2 ± 0.1 and a Schild slope of 1.1 ± 0.1. GSK256471 also produced a concentration-dependent rightward shift in the neurokinin B-concentration response curve (Fig. 2B) although, unlike GSK172981, a significant reduction in the maximal response (Emax) to neurokinin B was observed. Analysis according to the Schild equation (as described in Material and methods) yielded an empirical estimate of affinity (pA2 = 9.2 ± 0.2) The reversibility of the GSK256471-induced effect was examined by performing three washes following the application of GSK256471, before the addition of neurokinin B. The resulting antagonism profile (pA2 = 9.1 ± 0.1 with ~ 50% decrease in Emax) was not significantly different from the profile achieved in the absence of washout (data not shown).
3.4. Neurokinin B-induced neuronal firing in the guinea pig medial habenula The mean basal firing rate of neurokinin B sensitive neurones in the medial habenula in the GSK172981 (n = 16) and GSK256471 (n = 6) studies were determined as 5.3 ± 0.4 and 4.1 ± 0.6 Hz, respectively. The application of neurokinin B (10 nM–3 μM) produced concentrationdependent increases in neuronal activity with pEC50 values determined to be 6.9 and 7.0 for the GSK172981 and GSK256471 studies, respectively (Fig. 3A). GSK172981 and GSK256471 (both 100 nM) significantly reduced the spontaneous activity of medial habenula cells to 4.3 ± 0.5 Hz (n = 10, P = 0.0004) and 2.7 ± 0.5 Hz (n = 6, P = 0.013), respectively. In the presence of 100 nM GSK172981, there was a parallel rightward shift in the neurokinin B concentration response curve (Fig. 3A; apparent pKB = 8.1) with no significant effect on the maximal response (n = 12, P = 0.97; Fig. 3A). GSK256471 (100 nM) produced a rightward shift in the concentration–response relationship to neurokinin B but also reduced the normalised maximum attainable response from 1.41 ± 0.3 to 0.56 ± 0.1 (n = 5). Furthermore, in the presence of 1 μM GSK256471, the excitatory effect of neurokinin B (at up to 10 μM) was abolished (Fig. 3B). Quantitation of the shift of equi-effective neurokinin B concentrations (EC10) produced by 100 nM GSK256471
Fig. 3. The effect of (A) GSK172981 and (B) GSK256471 on the mean concentrationdependent increase in medial habenula neuronal firing elicited by neurokinin B in the guinea pig slice. Mean neuronal firing frequencies are expressed as a mean ± S.E.M. percentage of baseline (pre-drug; n = 5–6 per group).
using the Schild equation (Arunlakshana and Schild, 1959) yielded a pA2 value of 8.3.
3.5. Pharmacokinetic analysis in the rat and guinea pig The pharmacokinetic parameters of GSK172981 and GSK256471 in the rat and guinea pig following oral and intravenous administration are presented in Table 1. In addition, the comparative exposure and
Table 1 Pharmacokinetic evaluation of GSK172981 and GSK256471 in the rat and the guinea pig. Parameter
GSK172981
GSK172981
GSK256471
GSK256471
Species Doses
Rat i.v. 1 mg/kg p.o. 3 mg/kg
Guinea pig i.v. 1 mg/kg p.o. 3 mg/kg
Rat i.v. 1 mg/kg p.o. 3 mg/kg
Guinea pig i.v. 1 mg/kg p.o. 3 mg/kg
Blood clearance (ml/min/kg) Vss (l/kg) i.v. half life (t1/2) (h) Oral Bioavailability (%) Oral Cmax (ng/ml) Oral Tmax (h) p.o. half life (t1/2) (h) Doses (i.p.) Cmax (brain ng/g) Cmax (blood ng/ml) Brain:blood ratio
24 [23–25] 13 [9.2–16] 9.6 [6.912] 76 [69–82] 180 [146–219] 3 [2–4] ND
31 [26–35] 18 [16–19] 10 [8.6–12] 58 [53–63] 137 [136–138] 1 [1–1] ND
13 [9.0–15] 5.6 [4.9–6.7] 6.3 [5.5–7.1] 47 [40–51] 190 [179–204] 2.0 [2.0–2.0] ND
30 [27–34] 4 [3–4] 2 [1.8–1.9] 37 [24–47] 303 [230–417] 1 [1–1] 1.5 [1.5–1.5] 10 mg/kg (ip) 46 310 0.15
10 mg/kg (ip) 525 360 1.5
Data presented as mean (n = 2–3) per group and ranges of data [ ]. Intraperotineal (i.p.) dosing data are taken from pharmacological “wet dog shaking” behavioural assays n = 6–8 per group. Cmax— refers to the maximum concentrations achieved in blood and brain tissue; Vss — is volume of distribution; i.v. — intravenous administration; p.o. oral administration.
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brain penetration of both molecules following i.p. administration of a 10 mg/kg dose to the guinea pig are also shown. 3.6. Ex vivo occupancy of tachykinin NK3 receptors in guinea pig cerebral cortex As demonstrated by the decrease in ex vivo specific binding of the tachykinin NK3 receptor specific radioligand [3H]senktide, administration of both GSK172981 (1–30 mg/kg, i.p.) and GSK256471 (1– 100 mg/kg, i.p.) produced dose-dependent occupancy of NK3 receptors in guinea pig medial medial prefrontal cortex with estimated ED50 values of 0.8 and 0.9 mg/kg, respectively. Concurrent measurement of drug concentrations in hind brain and blood of the same animals revealed EC50 values of 52.5 ng/g and 39.2 ng/ml, respectively, for GSK172981 (Fig. 4A) and 8.0 ng/g and 39.2 ng/ml, respectively, for GSK256471 (Fig. 4B). 3.7. Senktide-induced guinea pig wet dog shake behaviours The i.c.v. administration of senktide significantly increased the occurrence WDS behaviour (per 30 min) from 2.4 ± 0.5 to 31.1 ± 5.1 (P < 0.05). Prior administration of GSK172981 (1–30 mg/kg, i.p.) significantly (F5,51 46.6, P < 0.05) attenuated senktide-induced WDS behaviour at (P < 0.05 for both 10 and 30 mg/kg doses) (Fig. 5A). GSK256471 significantly (F4,68 20.11, P < 0.0001) and dose-dependently attenuated the senktide-induced wet dog shake behaviour at both 10 and 30 mg/kg doses (P < 0.01 and P < 0.0001, respectively; Fig. 5B). 3.8. In vivo microdialysis: effects on haloperidol-induced increases in extracellular levels of dopamine in the guinea pig nucleus accumbens In the GSK172981 study, a significant effect of treatment was observed (F4, 32 7.3, P < 0.01; Fig. 6A). In this study, administration of haloperidol (0.5 mg/kg, s.c.) produced a significant (P < 0.001) increase in basal dopamine levels in the guinea pig nucleus accumbens reaching a maximum of 182 ± 14% of preinjection levels but had no effect on levels of noradrenaline or 5-HT (data not shown).
Fig. 5. Effects of (A) GSK172981 and (B) GSK256471 on senktide-induced “wet dog shaking” behaviours in the guinea pig. Data expressed as mean ± S.E.M. (n = 9–10) *P < 0.05, **P < 0.01, ***P < 0.001 versus senktide alone.
Pre-treatment with 30 mg/kg (i.p.) GSK172981 had no effect on basal levels of 5-HT, noradrenaline or dopamine but significantly (P < 0.05) attenuated the haloperidol-induced increase in nucleus accumbens dopamine concentrations when administered at 30 mg/kg (max. 131 ± 9.2%). Similarly, in the GSK256471 study, a significant effect of
Fig. 4. Relationship between ex vivo guinea pig medial prefrontal cortical tachykinin NK3 receptor occupancy and blood and brain exposures of (A) GSK172981 and (B) GSK256471 after i.p. dosing. Each point represents data from an individual animal.
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treatment was observed (F(6, 41) = 9.4390, P < 0.001; Fig. 6B). Again, haloperidol produced a significant increase in nucleus accumbens dopamine concentrations (max. 176 ± 10%; P < 0.001) which was significantly attenuated by the administration of 1 mg/kg (P < 0.05; max. 139 ± 4%), 3 mg/kg (P < 0.05; max 144 ± 17%) and 10 mg/kg (P < 0.001; max. 115 ± 13%) GSK256471. The highest dose of 30 mg/kg GSK256471, however, failed to attenuate the haloperidol-induced response. 4. Discussion Although dysfunction in several neurotransmitter systems has now been implicated in the pathophysiology schizophrenia, a cortical/ subcortical dopamine imbalance is still believed to be a core feature of the illness (Carlsson et al., 2001). Thus direct or indirect modulation of forebrain dopaminergic systems has remained a priority in the development of novel antipsychotics. Expression of tachykinin NK3 receptors in mesencephalic and mesolimbic dopamine pathways (Dam et al., 1990; Stoessl, 1994; Chen et al., 1998), together with the preclinical effects of receptor agonists (Bannon et al., 1986; Humpel et al., 1991; Keegan et al., 1992; Alonso et al., 1996) and small molecule antagonists (Nalivaiko et al., 1997; Gueudet et al., 1999; Dawson et al., 2008), all support a modulatory role for tachykinin NK3 receptors on dopaminergic neurotransmission and hence their potential therapeutic utility in schizophrenia. Recent evaluations of osanetant (Meltzer et al., 2004) and talnetant (Evangelista, 2005; Meltzer and Prus, 2006) in schizophrenia have further fuelled interest in tachykinin NK3 receptor antagonism as a novel antipsychotic mechanism, potentially with improved tolerability profiles compared with existing first and second generation antipsychotic drugs (Spooren et al., 2005). Unfortunately, these studies were not conclusive, possibly because the efficacy of these drugs may have been limited by their sub-optimal pharmacokinetic properties (Meltzer and Prus, 2006). In seeking molecules with improved
Fig. 6. Effects of (A) GSK172981 or (B) GSK256471 alone and on haloperidol mediated dopamine efflux in the guinea pig nucleus accumbens. Data expressed as mean ± S.E.M. (n = 8–12 per group). Both compounds alone produced no change in baseline dopamine. GSK172981 (panel A) produced an equipotent attenuation of the haloperidol-induced increase in extracellular dopamine for both 10 and 30 mg/kg doses (i.p.). GSK256471 (panel B) produced a dose related attenuation of the haloperidol-induced effect reaching a maximum reversal at 10 mg/kg (i.p.). However, 30 mg/kg returned to the haloperidol alone response i.e. was without affect demonstrating a “bell shaped” dose response.
characteristics, we now report the in vitro and in vivo pharmacological characterisation of two novel non-peptide tachykinin receptor antagonists, GSK172981 and GSK256471 (Smith et al., 2009). The comparative profile of the predecessor and clinical candidate molecule talnetant has been previously reported (SB223412; Sarau et al., 1997; Dawson et al., 2008). Both GSK172981 and GSK256471 exhibit high affinity and selectivity for the human tachykinin NK3 receptor (pKi values of 7.7 and 8.9, respectively). Furthermore, an evaluation of their functional profiles revealed significant divergence in their tachykinin NK3 receptor pharmacology. GSK172981 produced a parallel rightward shift in the neurokinin B concentration-dependent accumulation of IPx in human tachykinin NK3 receptor expressing U2OS cells, with no appreciable decrease in the maximal agonist response (Emax), a profile consistent with competitive antagonism. This competitive profile was also observed in a native tissue preparation in which medial habenula neuronal activity was recorded from guinea pig brain slices. Medial habenula neurons have a high sensitivity to tachykinin NK3 receptor agonists and an apparent absence of NK1 and NK2 receptor-mediated responses which makes this nucleus ideal for studies of central NK3mediated activity (Boden and Woodruff, 1994). GSK172981 produced a parallel rightward shift in the neurokinin B-induced neuronal excitations with no appreciable decrease in Emax, again indicative of competitive antagonist activity, with an apparent pKB of 7.8. In contrast, GSK256471 produced a rightward shift, but also a significant reduction in the maximal neurokinin B response in both the IPx accumulation assay (pA2 = 9.1) and in the native tissue electrophysiology assay. In the latter format, 1 μM GSK256471 completely abolished the neurokinin B response, but quantitation of the shift of equi-effective neurokinin B concentrations (EC10) produced by 100 nM GSK256471 using the Schild equation (Arulakshana and Schild, 1964) yielded a pA2 value of 8.3, which is comparable to its affinity as determined by radioligand binding studies. As with GSK172981, GSK256471 produces a concentrationdependent rightward shift in the neurokinin B response curve, but also causes a reduction in the maximal response, an effect that is not reversed by extensive washing of the cells prior to neurokinin B addition. This non-surmountable antagonist profile is consistent with agonist–antagonist hemi-equilibrium (Kenakin, 2004) which may result from a partially irreversible interaction with the receptor or a slow antagonist dissociation rate (Kenakin, 2004). The underlying reason for the difference in pharmacological profiles between these two structurally similar (Smith et al., 2009) molecules is unclear; further studies would be required to compare the kinetics of both compounds at the tachykinin NK3 receptor. GSK172981 exhibited good blood exposure following oral administration, with a calculated bioavailability of 76% in the rat, and markedly higher brain penetration (brain:blood ratio of 1.5 in the guinea pig) than either osanetant or talnetant (Dawson et al., 2008; brain:blood ratios in the guinea pig determined to be 0.3 for both compounds; osanetant data not shown). The good CNS penetration of GSK172981 was reflected in the reversal of tachykinin NK3 receptor agonist-induced wet dog shake behaviours (10 mg/kg, i.p.), a guinea pig pharmacodynamic model of central tachykinin NK3 receptor blockade (Renzetti et al., 1991; Yip and Chahl, 1997; Dawson et al., 2008), and ex vivo guinea pig medial medial prefrontal cortex tachykinin NK3 receptor occupancy which yielded an ED50 of 0.8 mg/kg and a brain Kd of ~50 ng/g. GSK256471 also achieved good systemic exposure following oral and i.p. dosing although its brain exposure was lower (guinea pig brain:blood ratio = 0.15). Nevertheless, despite the lower brain penetration, GSK256471 reversed senktide-induced wet dog shake behaviours (10 mg/kg, i.p.) and achieved 50% tachykinin NK3 receptor occupancy (0.9 mg/kg) at doses similar to GSK172981, although the latter was achieved at lower brain concentrations (Kd = 8 ng/g) due, presumably, to the higher affinity and potency of GSK256471.
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Differences in the pharmacological properties of small molecule ligands for tachykinin receptors across species is well documented (for review see Maggi, 1995) and greatly hampers the in vivo characterisation of novel therapeutics targeting this receptor. Generally lower affinity of tachykinin NK3 receptor antagonists such as PD154740, PD-157672 (Maggi, 1995), SR-142801 (Chung et al., 1995) and SB-223412 (Dawson et al., 2008) for rat and mouse versus human tachykinin NK3 receptors limits the value of rodent behavioural models conventionally used to identify antipsychotic-like potential. Native tissue antagonist radioligand binding assays revealed that the affinities of GSK172981 and GSK256471 for guinea pig cortical NK3 receptors (pKi values of 7.8 and 8.4, respectively) were comparable to those at the human receptor, making this a suitable species for further in vivo evaluation. Unfortunately, behavioural models designed to predict antipsychotic activity have not been established in this species. However, it is recognized that the activity of mesolimbic dopaminergic neurons is modulated by tachykinin NK3 receptors (Nalivaiko et al., 1997; Marco et al., 1998) and monitoring their output provides an in vivo means of evaluating tachykinin NK3 receptor mediated effects. In line with previous observations (Marco et al., 1998; Dawson et al., 2008; Gueudet et al., 1999), GSK172981 and GSK256471 had no effect on basal dopamine efflux in the nucleus accumbens, suggesting that NK3 receptors do not exert tonic activation of guinea pig dopaminergic neurons. However, stimulation of nucleus accumbens dopamine output by dopamine D2 receptor antagonism (Gueudet et al., 1999; Dawson et al., 2008), presumably achieved via the blockade of tonically active presynaptic autoreceptors, was attenuated by both GSK172981 and GSK256471. These data confirm and extend findings with talnetant (Dawson et al., 2008) and osanetant (Gueudet et al., 1999) and suggest that tachykinin NK3 receptor antagonism can reduce hyperactivity in the mesolimbic dopamine system. GSK172981 appeared more potent in reversing the haloperidol-induced dopamine efflux in the nucleus accumbens than the previous molecule talnetant (Dawson et al., 2008), presumably reflecting the improved brain penetration of this molecule. GSK256471 exhibited an even greater enhancement in in vivo potency and was effective at the lowest dose tested (i.e. 1 mg/kg). Furthermore, whereas the GSK172981 produced a talnetant-like partial reversal of the haloperidol-induced dopamine efflux (Dawson et al., 2008), GSK256471 fully attenuated dopamine levels back to baseline. The explanation for the apparent bell-shaped dose response to GSK256471 in this paradigm is not clear, but the relative low CNS exposure together with a good selectivity profile would suggest that this is unlikely to be an “off target” mediated effect. Comparing these datasets can provide some potential insight into functional differences between these two molecules in vivo. GSK172981 appeared to require tachykinin NK3 receptor occupancy in excess of 90% to reverse haloperidol stimulated dopamine efflux. This value is similar to that reported for the predecessor competitive tachykinin NK3 receptor antagonist talnetant (Dawson et al., 2008). Whilst GSK256471 was effective at a dose that equated to an occupancy of only 50%, possibly reflecting the different pharmacologies of the two molecules i.e. the competitive antagonism of GSK172981 requiring relatively higher levels of receptor occupancy for in vivo efficacy, whereas the apparent non-surmountable pharmacology of GSK256471 appearing to generate efficacy at lower levels of receptor occupancy. A similar phenomenon has also been observed with molecules targeting the NK1 receptor (Lindström et al., 2007). Thus if this phenomena were to occur in human then one may speculate that the circulating trough levels required for efficacy in the clinic would need to be significantly lower for GSK256471 cf. GSK172981. In this regard, it is noteworthy that osanetant exhibits a non-surmountable pharmacology at tachykinin NK3 receptors in vitro (Emonds-Alt et al., 1995) appeared to be clinically efficacious at very low Ctrough plasma concentrations (Meltzer and Prus, 2006), possibly substantiating this hypothesis. However, there are a number
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of potential technical caveats to this quite speculative hypothesis and thus this cannot be truly substantiated until these molecules enter the clinic. In conclusion, these results provide further preclinical support for the utility of tachykinin NK3 receptor antagonism in the treatment of schizophrenia and that the novel tachykinin NK3 receptor antagonists GSK172981 and GSK256471 exhibit in vivo potency superior to that of talnetant (Dawson et al., 2008) and hence may have potential as novel antipsychotic drugs. References Alonso, R., Fournier, M., Carayon, P., Petitpretre, G., Le Fur, G., Soubrie, P., 1996. Evidence for the modulation of dopamine-neuronal function by tachykinin NK3 receptor stimulation in gerbils. Eur. J. Neurosci. 8, 801–808. Ames, R., Fornwald, J., Nuthulaganti, P., Trill, J., Foley, J., Buckley, P., Kost, T., Wu, Z., Romanos, M., 2004. BacMam recombinant baculoviruses in G protein-coupled receptor drug discovery. Recept. channels 10, 99–107. 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