NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-β-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-β-Lactam That Modulates Synaptic... Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo- 2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-β-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for “off-target” activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100–500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01–1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders. Keywords: NMDA receptor, learning and memory, synaptic plasticity Received: August 2, 2017; Revised: October 7, 2017; Accepted: October 17, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any 242 medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 243 Significance Statement NYX-2925 is a novel NMDA receptor-specific modulator that facilitates synaptic plasticity and has therapeutic potential for a v - ar iety of NMDA receptor-mediated central nervous system (CNS) disorders. NYX-2925 was synthesized using a novel spirocyclic-β- lactam chemical approach using rapastinel (formerly GLYX-13) as a template, which was in turn synthesized from a hypervariable region of a unique monoclonal antibody with NMDA receptor modulatory properties. Thus, the creation of NYX-2925 completes the process of developing monoclonal antibodies to help elucidate the molecular mechanisms of complex biological processes such as learning and memory and converting them into small molecules with therapeutic potential. Introduction N-methyl-D-aspartate (NMDA) receptors are one of a family of and activity profiles. NYX-2925 is a representative compound ligand gated ionotropic glutamate receptors that are found pre- from this platform. Figure  1 shows the structure of rapastinel dominantly in the CNS and developmentally regulated (Cull- and NYX-2925, and we report on its pharmacological, toxico- Candy et al., 2001; Traynelis et al., 2010). They are unique among logical, functional, and mechanistic properties. glutamate receptors in that they require both glutamate and glycine for full activation (Danysz and Parsons, 1998). They are Materials and Methods heterotetrameric complexes that are expressed as multiple sub- types each with unique properties (Paoletti et al., 2013). NMDA Animals receptors play a pivotal role in modulating normal neuronal functions including activity-dependent synaptic plasticity asso- Adult male Sprague-Dawley rats from Harlan or Charles River ciated with learning and memory (Bliss and Collingridge, 1993; were used for most studies. For the novel object recognition Yashiro and Philpot, 2008; Morris, 2013) and have been impli- study, adult male Lister Hooded rats from Harlan were used. cated in a variety of CNS disorders, including schizophrenia Rats were group housed (3–4 per cage) in Lucite cages with aspen (Coyle, 2012; Goff, 2012), mood disorders (Ghasemi et  al., 2014; wood chip bedding, maintained on a 12-hour-light/12-hour-dark Vasilescu et  al., 2017), epilepsy (Ghasemi and Schachter, 2011), cycle (lights on at 5:00 am), and given ad libitum access to Purina neuropathic pain (Millecamps et  al., 2007Zhou ; et  al., 2011), Lab Chow and tap water throughout the study. For the drug dis- fibromyalgia (Harris et al., 2008; Pyke et al., 2016), Rett syndrome crimination study, rats were singly housed with ad libitum access (Patrizi et  al., 2016), and cognitive decline due to normal aging to water. All experiments were approved by the Northwestern (Robb, 1991; Burgdorf et al., 2011a) among others. University, Virginia Commonwealth University, and New York Recently, reports have described positive clinical trial Medical College Institutional Animal Care and Use Committees. data with mechanistically distinct NMDA receptor modula- tors including rapastinel (formerly GLYX-13) and ketamine for Drugs depression (Fond et al., 2014; Preskorn et al., 2015) and obsessive- compulsive disorder (Rodriguez et al., 20132016 , ), D-cycloserine NYX-2925 was synthesized by Sai Life Sciences (India) and was for schizophrenia (Cain et  al., 2014) and posttraumatic stress administered p.o. (0.1–10  mg/kg) in 1  mL/kg in 0.5% carboxy- disorder (de Kleine et al., 2012), and memantine for Alzheimer’s methylcellulose (CMC) 0.9% sterile saline. The NMDA receptor disease (Wilkinson et al., 2014). The key role that NMDA recep- glutamate site antagonist CPP ((±)-3-(2-carboxypiperazin-4-yl) tors play in synaptic plasticity throughout the CNS, the marked propyl-1-phosphonic acid) was purchased from Sigma and increase in NMDA receptor mechanistic studies, including X-ray administered i.p. (10  mg/kg) in 1  mL/kg 0.9% sterile saline. The crystallographic analysis (Karakas and Furukawa, 2014 Dolino ; dose of CPP (10 mg/kg i.p.) was chosen based on previous reports et  al., 2015; Lu et  al., 2017), and biophysical studies on recep- that this dose could block the antidepressant-like effects of an tor subtype properties (Tavoloni and Schaffner, 1989 Iacobucci ; NMDAR positive modulator without exhibiting behavioral effects and Popescu, 2017), coupled with clinical trial successes seen on its own (L. Zhang et  al., 2013; Burgdorf et  al., 2015b). The with NMDA receptor modulators, make this receptor complex 5-HT receptor antagonist SB399885 was purchased from GVK an attractive target for drug discovery. Biosciences (India) and was administered p.o. (10 mg/kg) in 2 mL/ Rapastinel is a tetrapeptide (threonine-proline-proline- kg 1% CMC and was used as a positive control in the novel object threonine) derived from a hypervariable region of a monoclonal recognition study (Hirst et al., 2006). Ketamine-HCl (Ketalar) was antibody B6B21 (Moskal et al., 2005). B6B21 was shown to act as obtained from Patterson Veterinary Inc. and was diluted with a cognitive enhancer (Thompson et  al., 1992) with glycine-site 0.9 % saline to the concentration required to provide the desired partial agonist properties at the NMDA receptor (Haring et  al., dose in a 1-mL/kg volume for both i.p. and p.o. administration. 1991). Rapastinel has also been found to be a robust cognitive enhancer with marked antidepressant-like effects in a variety of [ H] MK-801 Potentiation Assay rat models (Burgdorf et al., 2013 2017 , ). Mechanistically, rapasti- nel appears to bind directly to NMDA receptors, triggering an NMDAR Subtype Expressing HEK Cell Membrane Preparation increase in AMPA receptor activity and leading to a long-term Crude membranes were prepared using transiently transfected, potentiation-like increase in synaptic plasticity associated with NMDAR-expressing HEK cells, described below. All procedures learning (Moskal et al., 2017). were performed at 4°C. Briefly, pelleted cells were initially A key structural feature of rapastinel is its dipyrrolidine- washed in 10 mM Tris acetate (pH 7.4 at 4°C), pelleted, and fro- based β-turn motif. A novel chemical platform was created using zen at -80°C overnight. The pellet was then resuspended and spirocyclic-β-lactam chemistry (Bittermann and Gmeiner, 2006) homogenized (30 strokes) in a glass homogenizer and pelleted with a variety of NMDA receptor subtype selectivity, potency, at 51500 x g for 30 minutes at 4°C and stored at –80ºC until assay. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 244 | International Journal of Neuropsychopharmacology, 2018 Figure  1. Structural comparison of the peptide rapastinel with the spirocyclic β-lactam, ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl) butanamide) (NYX-2925). Spirocyclic β-lactam chemistry has been used to create a number of new molecules that mimic key properties of their parent peptides (Bittermann and Gmeiner, 2006). Functional glycine site agonist effects were measured using Bioavailability an [ H] MK-801 potentiation assay. Briefly, 300 µg of membrane Male Sprague Dawley rats were dosed with NYX-2925 (1 mg/kg extract protein were preincubated for 15 minutes at 25°C in the p.o.), and jugular vein blood draws taken in K2-EDTA-treated presence of a saturating concentration of glutamate (50  µM) and tubes and cisterna magna cerebrospinal fluid (CSF) draws were varying concentrations of NYX-2925. Following the addition of taken at various time points. Samples were maintained at 4°C 0.3mCi [ H] MK-801 (Amersham, 22.5 Ci/mmol), reactions were for 30 to 60 minutes after collection and stored at 80°C until incubated for an additional 15 minutes (nonequilibrium con- assay. On the day of the assay, plasma, CSF, and standards were ditions). Bound and free [H] MK-801 were separated via rapid thawed at 4°C. Samples were extracted with acetonitrile and filtration. Zero levels were determined in the absence of any NYX-2925 levels were assessed by liquid chromatography tan- glycine ligand. The percent maximal [H] MK-801 binding was dem mass spectrometry and the lower limit of quantification for calculated relative to stimulation measured in the presence of this assay was ~4 nM. 1 mM glycine and 50 µM glutamate. Binding curves were fitted using GraphPad software. Dendritic Spine Morphology Analysis Creation of the Stable hNMDAR1-Expressing HEK Dendritic spine analyses were conducted as previously described Cell Line (Ota et  al., 2014; Burgdorf et  al., 2015b) using the Afraxis ESP The cDNA encoding the human GluN1-1 (GenBank BC156961) Platform (Afraxis, Inc.). Animals were given a single dose of was amplified from MGC clone 100063609 using pfu polymerase NYX-2925 (1 mg/kg p.o.), or 0.5% Na-CMC in 0.9% sterile saline and subcloned into the pCMV/zeoDNA3 vector using standard vehicle (1  mL/kg), and 24 hours post dosing they were deeply molecular techniques and verified by direct sequencing. HEK anesthetized (isoflurane) and brains fixed via cardiac perfusion cells (ATCC) were transfected with the mutant construct using using 4% paraformaldehyde. Brains were stored in ice cold 0.1 X-tremeGENE 9 transfection reagent (Roche) and stable clones M phosphate buffer and stored at 4°C until sectioning. Brains selected in Zeocin-containing media. were sectioned using a Vibratome (Leica VT1000) to collect sec- tions (300 μm thick) from the anterior to posterior extremes of each brain. Ballistic dye labeling (DiI and DiO; 3  mg dissolved Creation of the Transient hNMDAR2 (A-D) in methylene chloride and coated on tungsten particles) was Expression Vectors performed using a commercially available gene gun (Bio-Rad) cDNAs encoding the hNMDAR2A (GENBANK #NM_000833, to label neurons. Thick sections were mounted to slides with pfu polymerase amplified from human cortex cDNA), hNM- raised barriers using ProLong Gold (Life Technologies) and cover DAR2B (GENBANK #NM_000834, pfu polymerase amplified slipped. Laser-scanning confocal microscopy (Olympus FV1000) from Open Systems clone #8322670), hNMDAR2C (GENBANK was performed using a 63 × objective (1.42 NA) to scan individu- #NM_000835, pfu polymerase amplified from OriGene clone ally labeled neurons at high resolution (0.103 × 0.103 × 0.33  μm #SC300138), and hNMDAR2D (GENBANK #NM_000836, pfu voxels). Microscopy was performed blind to experimental condi- polymerase amplified from OriGene clone #SC300139) were tions. A minimum of 5 cells per animal were sampled. Primary subcloned into the pCMV6/XL5 vector using standard mo- dendrites within the inner molecular layer were analyzed, and lecular techniques. samples (50 μm) were collected from primary dendrites starting at 100 μm from the soma. Spine head and neck sizes were ana- lyzed given that larger spine head and necks are associated with Off-Target Receptor Binding Assays greater NMDAR-mediated calcium flux (Noguchi et al., 2005) as To further characterize the target specificity of NYX-2925, we well as the induction of LTP (Matsuzaki et al., 2004). tested the ability of 10  μM NYX-2925 (>10 000 × Cmax) to com- Blind deconvolution (AutoQuant) was applied to raw 3‐ pete for binding in a radioligand displacement assay using a dimensional digital images that were then analyzed for spine broad panel of known CNS protein targets (LeadProfilingScreen density and morphology by trained analysts. Individual spines 2, Eurofins Cerep). The binding panel included adenosine were measured manually for (a) head diameter, (b) spine and adrenergic receptor sites; dopamine, histamine, and opi- length, and (c) spine neck diameter from image Z-stacks using oid receptors; calcium channels; and muscarinic cholinergic software custom-designed by Afraxis Inc. Each dendrite was receptor sites. analyzed by 3 to 4 independent analysts. Analysts were blinded Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 245 to all experimental conditions (including treatment, brain stimulation induction. NYX-2925 was bath applied for 40 to region, and cell type). 50 minutes starting 20 minutes before application of high-fre- quency stimulation. Extracellular Hippocampal Recordings Metaplasticity Studies Experiments were conducted as described previously (Burgdorf Experiments were conducted as described previously (Zhang et  al., 2015b). Rats were dosed with NYX-2925 (1  mg/kg p.o.) et al., 2008; Burgdorf et al., 2015b). Adult male rats were deeply and hippocampal slices were prepared either 24 hours or 1 anesthetized with isoflurane and decapitated. Brains were week post dosing. Three submaximal bouts of high-frequency removed rapidly, submerged in ice-cold artificial CSF (ACSF, Schaffer collateral stimulation (2 × 100 Hz/800  ms) were applied 2–4°C), which contained (in mM): 124 NaCl, 4 KCl, 1.5 MgCl, 2.5 20 minutes apart. LTP was measured 40 minutes after the last CaCl , 1.25 NaH PO , 26 NaHCO , 10 glucose; at pH 7.4, gassed 2 2 4 3 high-frequency bout of stimulation. continuously with 95% O/5% CO . Brains were hemisected, the 2 2 frontal lobes removed, and individual hemispheres glued using cyanoacrylate adhesive onto a stage immersed in ice-cold ACSF Intracellular Recordings from Hippocampal CA1 gassed continuously with 95% O/5% CO during slicing. Then 2 2 Pyramidal Neurons 400-μm-thick coronal slices were cut using a Vibratome (Leica Whole-cell patch clamp recordings from CA1 pyramidal neurons VT1200S) and transferred to an interface holding chamber for were acquired as described previously (Burgdorf et  al., 2013). incubation at room temperature for a minimum of 1 hour be- Patch pipette resistance was 6 to 6.5 MΩ when filled with intra- fore transferring to a Haas-style interface recording chamber cellular solution that contained (in mM): 135 CsMeSO , 8 NaCl, continuously perfused at 3  mL/min with oxygenated ACSF at 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 Na-GTP, and 1 QX-314 [N-(2,6- 32 ± 0.5°C. dimethylphenylcarbamoylthyl)-triethylammonium bromide], Low resistance recording electrodes were made from thin- 275 mOsm, pH 7.25 adjusted with Cs(OH) . CA1 pyramidal neu- walled borosilicate glass (1–2 MΩ after filling with ACSF) and rons were visualized by infrared imaging and patched using a inserted into the apical dendritic region of the Schaffer collat- 60x water-immersed objective mounted to a Zeiss microscope eral termination field in stratum radiatum of the CA1 region (Axioskop 2 Fs plus). After whole-cell voltage clamp configura- to record field excitatory postsynaptic potentials (fEPSPs). A bi- tion was established, access resistance was carefully monitored, polar stainless-steel stimulating electrode (FHC) was placed on and only cells with stable access resistance (<5% change) were Schaffer collateral-commissural fibers in CA3 stratum radiatum, included in analyses. Excitatory postsynaptic currents (EPSCs) and constant current stimulus intensity adjusted to evoke ap- were recorded using a MultiClamp 700B (Molecular Devices); proximately half-maximal fEPSPs once each 30 seconds (50–100 with the low-pass filter setting at 1 to 3 kHz, series resistance pA; 100-μs duration). fEPSP slope was measured before and after was compensated in the voltage-clamp mode, and patched cells induction of LTP or LTD by linear interpolation from 20% to 80% whose series resistance changed by >10% were rejected from of maximum negative deflection, and slopes confirmed to be analysis. Signals were filtered at 3 kHz and digitized at 10 kHz stable to within ± 10% for at least 15 minutes before commencing with a Digidata 1322A controlled by a Clampex (v9.2) (Molecular an experiment. Signals were recorded using a Multiclamp 700B Devices). A  bipolar tungsten stimulating electrode (FHC) was amplifier and digitized with a Digidata 1322 (Axon Instruments). placed in the Schaffer collateral-commissural fibers in CA3 Data were analyzed using pClamp software (version 9, Axon stratum radiatum and stimulus pulses (800  µS duration) were Instruments). delivered at 15- to 30-second intervals. Neurons were voltage clamped at -70 mV to record EPSCs to assess input-output rela- Hippocampal LTP and LTD Studies tions and paired-pulse facilitation. Neurons were clamped at -40 Experiments were conducted as described previously (Zhang mV for recording NMDA currents to relieve voltage-dependent et al., 2008). LTP was induced by 2 theta trains pared by 3 min- magnesium block, and slices were perfused with ACSF contain- utes (3× 100 Hz/500  ms) and LTD induced by a low-frequency ing 0 added magnesium, 3  mM calcium, 10  µM picrotoxin, and stimulus train (2 Hz/10 min). LTP/LTD was measured 40 minutes 10 µM CNQX to isolate NMDA conductances. after high- or low-frequency stimulation. NYX-2925 was bath All recording pipette solutions were made with deionized applied for 40 to 50 minutes starting 20 minutes before applica- −2 distilled water (resistance >18 M Ω cm ; Milli-Q system). Data tion of high- or low-frequency stimulation. were analyzed initially with Clampfit (v9) (Axon Instruments) and further processed and presented with Origin 6.1 (Microcal Medial Prefrontal Cortex (MPFC) LTP Studies Software) and CorelDraw 10.0 (Corel) programs. Experiments were conducted as described previously (Burgdorf et  al., 2015a). Four-hundred-μm thick slices were cut using a vibratome (Leica VT1200S) in a modified coronal orientation Novel Object Recognition (NOR) (Parent et  al., 2010) containing both prelimbic and infralimbic regions of the MPFC that are targets (via the fornix) of the hip- Experiments were conducted as described previously (Hirst et  al., 2006). Rats were habituated to the NOR test box twice a pocampal-MPFC pathway. Recording electrodes were inserted into layer III/IV of the prelimbic MPFC and monosynaptically day for 2 consecutive days prior to testing. Each habituation ses- sion was comprised of a 3-minute exposure to the empty test evoked fEPSPs evoked by mixed excitatory inputs were recorded from layer V pyramidal neurons. A bipolar tungsten stimulating box (46 x 30 x 45  cm), followed by 1 minute in the side annex (1 x 30 x 45 cm) and a further 3 minutes in the test box, thereby electrode (FHC) was placed on MPFC deep white matter. LTP was induced by stimulation of input axons with 3 high-frequency mimicking the test protocol. A  vehicle dose was administered prior to one of the habituation sessions on each habituation day. theta burst stimulus trains of 10  ×  100 Hz/5 pulse bursts each, applied at an inter-burst interval of 200 milliseconds. Each train The NOR test comprised 2 test sessions, T1 and T2, each last- ing 3 minutes. On the first test day, T1, rats were habituated to was 2 seconds in duration, and trains were applied 3 minutes apart. LTP/LTD was measured 40 minutes after high-frequency the empty test box for 3 minutes and then placed in the side Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 246 | International Journal of Neuropsychopharmacology, 2018 annex for approximately 1 minute whilst 2 identical test objects minutes using a within-subjects design. Habituation and test- were placed in the test arena equally spaced to each other and ing consisted of placing rats onto the fixed speed version of the the side walls. The rat was then returned to the test arena and Rota-Rod test (16 RPM) for 300 seconds, and the latency to fall off allowed to freely explore the objects for a further 3 minutes. At the Rota-Rod was recorded. the end of the test session, the rat was returned to its home cage. Following a 24-hour inter-trial interval, the recall trial (T2) Open Field Test of Locomotor Activity was conducted. T2 was similar to T1 except that one of the “fa- Open field testing was conducted as previously described miliar” objects was substituted for a novel one of a similar size (Burgdorf et al., 2013). Rats were placed into the center chamber of and color but different shape. The objects used were made of the open field (40 x 40 x 20 cm) for 10 minutes under dim-red light- black hardened plastic and were geometric shapes (towers and ing 1 hour post dosing with NYX-2925 (1  mg/kg p.o.) or vehicle pyramids) that were of no relevance to the animals. Objects were (1 mL/kg p.o.). Line crosses were scored offline from video record- cleaned between trials with 70% ethanol to eliminate odor cues. ings by an experimenter blinded to the treatment condition. T1 and T2 trials were recorded by GeoVision surveillance camera software and files were saved on DVD for remote scor- ing by an operator blinded to the treatments. Exploration was Drug Discrimination scored as time spent sniffing or licking the objects, when the Testing was conducted as described previously (Burgdorf nose was in contact with the object and was moving (i.e., when et  al., 2013). Adult male Sprague-Dawley rats were trained to the animal was sniffing). Sitting on the object or next to it with discriminate ketamine (5.6 mg/kg, i.p.) from saline (1 mL/kg, i.p.) the nose directed away was not classed as exploration. The dis- 5  d/wk (Monday-Friday; 15  min/d) in standard 2-lever operant crimination index [(time spent exploring the novel object - time conditioning chambers (Med Associates) under a double-alter - spent exploring the familiar object)/total exploration time] was nation schedule of ketamine or saline (K, K, S, S, K, K, etc.). Rats then calculated for T2. were placed in the operant conditioning chamber 10 minutes For the NYX-2925 groups, 0.01, 0.1, or 1  mg/kg p.o. was post-injection and the session initiated as signaled by illumina- administered 60 minutes before T1 and CMC saline adminis- tion of the chamber house light. Completion of 10 consecutive tered 1 hour before T2. For the SB399885 group, 10  mg/kg p.o. responses (fixed ratio 10; FR 10) on the correct lever resulted in was administered 240 minutes before both T1 and T2. For the ve- delivery of a 45-mg food pellet (Bio-Serv) and illumination of a hicle group, CMC saline (1 mL/kg) was administered 60 minutes white stimulus light over the lever. Incorrect responding reset before both T1 and T2. the FR for correct-lever responding. Food (Harlan Teklad Rodent Diet) access beyond those obtained during behavioral ses- Ultrasonic Vocalization (USV) Assay sions was restricted to ~20 g given post session. Animals were tested following administration of various doses of NYX-2925 Heterospecific rough-and-tumble play was conducted as pre- (1, 10, 100 mg/kg, p.o.), CMC saline (2 mL/kg) or ketamine (10, 17 viously described (Burgdorf et  al., 2011b). The experimenter 30 mg/kg, p.o.), and lever selection and rates of responding were was blind to the treatment condition of the animals. Animals recorded. Doses of NYX-2925 and its vehicle were administered received 3 minutes of heterospecific rough-and-tumble play 1 hour prior to the session start and p.o. ketamine was adminis- consisting of alternating 15-second blocks of heterospecific tered 15 minutes prior to session start. For all p.o. test sessions, play and 15 seconds of no stimulation. High-frequency USVs rats were also administered i.p. saline, 1 mL/kg, 10 minutes prior were recorded and analyzed by sonogram in a blind manner to session start to mimic training conditions. Training contin- as described previously (Burgdorf et  al., 2011b). At the end of ued under the double alternation of 5.6  mg/kg ketamine and the 3-minute session, the latency for the rat to approach the saline injections between test sessions. Illumination of lights, experimenters hand to self-administer heterospecific play was recording of responses, and pellet delivery were performed with also measured. Animals were not habituated to play stimula- MED-PC operant conditioning software (version 1.1). tion before dosing and testing. Using this paradigm, the in- crease in 50-kHz USVs that occurs across trial blocks reflects positive emotional learning (Burgdorf et  al., 2011b Ishiy ; ama Statistical Analysis and Brecht, 2016). Behavioral and electrophysiological data were analyzed by Animals were administered NYX-2925 (1 mg/kg p.o.) or CMC ANOVA, followed by Fisher’s PLSD posthoc test (Statview). The saline (p.o.) 1 hour before testing. Rats received the NMDA re- level of statistical significance was set at P < .05. ceptor glutamate site antagonist CPP (10  mg/kg i.p.) or sterile Data from the [H] MK-801 potentiation assay data was ana- saline (i.p.) 2 hours before NYX-2925 (1 mg/kg p.o.) or CMC sa- lyzed by Prism (Graphpad). line (p.o.). This dose of NYX-2925 was chosen given that it leads to CSF exposures that activate all 4 NMDAR subtypes (Figures 2A-D and 4A) and showed maximal facilitation of learning and Results memory in the novel object recognition test (Figure 5A). [ H] MK-801 Potentiation and Off-target Receptor Rota-Rod Test of Motor Coordination Binding Rota-Rod testing was conducted as previously described NYX-2925 facilitated [H] MK-801 binding in all four human (Nadeson et al., 2002) using a 4-station Rota-Rod apparatus (Med NMDAR2 subtypes. The concentration-response curves for NYX- Associates). One day before testing, animals received 3 Rota-Rod 2925 assessed in hNR1-expressing HEK cells transfected with habituation sessions with at least 30 minutes between each ses- hNR2A, 2B, 2C, and 2D are shown in Figure 2. The activity of NYX- sion and an additional habituation session immediately before 2925 was 40.6%, 47.1%, 63.1%, and 57.8% of [ H] MK-801 activity dosing (0 min). Animals were dosed with NYX-2925 (1–100 mg/ measured in the presence of maximal glycine for hNR2A, 2B, 2C, kg p.o.) or CMC saline. Animals were tested 15, 30, 60, and 120 and 2D receptors, respectively. The potency (EC) of NYX-2925 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 247 Figure 2.NYX-2925 is active at all 4 human N-methyl-D-aspartate receptor (hNMDAR) 2 subtypes. Potentiation of [ H] MK-801 binding by NYX-2925 and glycine in hNM- DAR subtype-expressing human embryonic kidney (HEK) cells. Stable hNMDAR1-expressing HEK cells were transiently transfected with cDNAs encoding hNMDAR2A, hNMDAR2B, hNMDAR2C, or hNMDAR2D. At 48 hours post-transfection, membrane-bound receptors were isolated, and the functional glycine site agonist effects were measured using the [ H] MK-801 potentiation assay, as described in Methods. The percent maximal effects were calculated relative to stimulation in the presence of 1 mM glycine and 50 μM glutamate. Binding curves were fitted using GraphPad Prism software. was 55 pM, 28 fM, 11 pM, and 55 pM for hNR2A, 2B, 2C, and 2D [5000 nM] vs vehicle, P < .05; Figure  3C). Moreover, LTP at mixed receptors, respectively. excitatory inputs synapsing in layer II/III of MPFC was also facili- NYX-2925 (10 µM) did not exhibit any significant affinity (con- tated by NYX-2925 (F = 5.6, P < .05; Fisher’s PLSD posthoc test (4,27) sidered >50%) for any of the 81 binding sites tested (Table). 1 At 10 NYX-2925 [100 and 500 nM] vs vehicle, P < .05; Figure 3D). μM, NYX-2925 did not demonstrate significant inhibition or stim- ulation (agonism) in any of the 81 sites. This study provides evi- Pharmacokinetics and Toxicology dence that NYX-2925 has a low potential for “off-target” activity. NYX-2925 showed high oral bioavailability, CNS penetration, and had a wide therapeutic index. As shown in Figure 4A, NYX-2925 NMDAR Current and Long-Term Potentiation in (1 mg/kg p.o.) has a plasma Cmax of 706 nM at 1 hour, a plasma Hippocampus and Medial Prefrontal Cortex half-life of 6.8 hours, a CSF Cmax of 44 nM at 1 hour, and a CSF NYX-2925 (100–500 nM) increased the magnitude of pharmaco- half-life of 1.2 hours. The Cmax CSF exposure is 44 nM. The oral logically isolated NMDA current in Schaffer collateral-evoked bioavailability of NYX-2925 in plasma is 56% as calculated by the EPSCs in CA1 pyramidal neurons (repeated-measures ANOVA area under the curve of plasma exposure following 2 mg/kg i.v. F = 7.2, P < .05; within-subjects t test vs baseline NYX-2925 or 10 mg/kg p.o. dosing (mean ± SEM AUC IV 2155.12 ± 45.77, p.o. (3,18) [100 and 500 nM] vs vehicle, P < .05; Figure  3A). NYX-2925 also 6053.73 ± 498.55 ng.h/mL). increased the magnitude of LTP at Schaffer collateral-CA1 syn- The no observed adverse effect level of NYX-2925 for male apses (F = 6.2, P < .05; Fisher’s PLSD posthoc test NYX-2925 [500 Sprague Dawley rats when administered by the oral (gavage) (4,30) nM] vs vehicle, P <.05; F igure 3B) and inhibited LTD at these same route, once daily for 14 consecutive days is 1000  mg/kg/d as synapses (F = 4.1, P < .05; Fisher’s PLSD posthoc test NYX-2925 measured by mortality, clinical signs, body weight, food intake, (3,28) Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 248 | International Journal of Neuropsychopharmacology, 2018 Table 1. Radioligand Displacement by NYX-2925 (10 μM) Table 1. Continued NYX-2925 NYX-2925 Site % Inhibition Site % Inhibition A1 (antagonist) -4.2 5-HT2C (antagonist) -5.9 A2A (agonist) -6.1 5-HT3 (antagonist) 2.7 A3 (agonist) -4.9 5-HT5a (agonist) -7.5 α1 non-selective (antagonist) -10 5-HT6 (agonist) -8.6 α2 non-selective (antagonist) -11.6 5-HT7 (agonist) -5.1 β1 (agonist) 4.6 Sigma (non-selective) (agonist) -15.8 β2 (agonist) -7.6 SSST (non-selective) (agonist) -13.8 AT1 (antagonist) -0.8 GR (agonist) -6.2 AT2 (agonist) 1.8 VPAC1 (VIP1) (agonist) 7.4 BZD central (agonist) -9.1 V1 a (agonist) 15.3 BZD peripheral (antagonist) -6.6 Verapamil site (antagonist) 3.4 BB non-selective (agonist) -9 hERG [3H] Dofetilide -3 B2 (agonist) -3.1 KV channel (antagonist) -7.4 CGRP (agonist) 3.6 SKCa channel (antagonist) 5 CB1 (agonist) 5.7 Na+ channel (site 2) (antagonist) 7.3 CCK1 CCKA (agonist) 3.8 Cl- channel GABA-gated (antagonist) 5.2 CCK2 CCKB (agonist) -13 Norepinephrine transporter (antagonist) -6.2 D1 (antagonist) -19.9 Dopamine transporter (antagonist) -6.4 D2S (antagonist) 6.8 5-HT transporter (antagonist) -13.9 D3 (antagonist) -5.2 Results that show an inhibition or stimulation >50% D4.4 (antagonist) -5 are considered to represent significant effects of the test compound. D5 (antagonist) 4.1 ETA (agonist) 5.7 ETB (agonist) 1.3 and gross histopathology as well as organ weights. The plasma GABA non-selective (agonist) -18.8 exposure (area under the curve) of NYX-2925 following 14 daily GAL1 (agonist) 3 doses of 1000  mg/kg was 1 074 478  ng.h/mL compared with GAL2 (agonist) 3.7 494 ng.h/mL for a single behaviorally efficacious dose of 1 mg/ PDGF (agonist) -2.6 kg, thus resulting in a projected therapeutic index of 2175. CXCR2 IL-8B (agonist) -4.5 CCR1 (agonist) 0 Structural Plasticity TNF-α (agonist) -21.7 H1 (antagonist) 2.4 NYX-2925 (1  mg/kg p.o.; 24 hours post dosing) induced struc- H2 (antagonist) 0.5 tural plasticity as indexed by increased diameter of spine heads MC4 (agonist) 3.4 (F = 44.2, P < .05) and necks (F = 27.2, P < .05) in the pri- (1,3441) (1,3441) MT1 ML1A (agonist) 1.4 mary apical dendrites of dentate granule neurons (Figure  4B). M1 (antagonist) -23.4 Spine length (F = 2.9, P > .05) or total spine number (F = 1.1, (1,3441) (1,65) M2 (antagonist) -6.1 P > .05) was not affected by NYX-2925 (Figure 4B). M3 (antagonist) -10.1 M4 (antagonist) -11.3 M5 (antagonist) 5.6 Metaplasticity NK1 (agonist) -22.5 NYX-2925 persistently enhanced LTP at 24 hours and 1 week NK2 (agonist) 2.4 following a single dose (1 mg/kg p.o.) at Schaffer collateral-CA1 NK3 (antagonist) -7.7 synapses after 3 submaximal high-frequency stimulus trains Y1 (agonist) -3.4 Y2 (agonist) -9.9 (2 × 100 Hz/800  ms, arrows). LTP was evaluated either 24 hours NTS1 / NT1 (agonist) -11.4 post dosing (F = 5.5, P < .05) or 1 week post dosing (F = 8.8, (1,13) (1,14) δ2 (DOP) (agonist) -4.2 P < .05) as shown in Figure 4C-D. κ (KOP) (agonist) 0.1 μ (MOP) (agonist) -4.5 Learning and Memory Tests NOP / ORL1 (agonist) 5.9 PAC1 / PACAP (agonist) -4.4 NYX-2925 facilitated learning and memory in multiple para- PPARγ (agonist) -10.7 digms across a wide dose range. NYX-2925 (0.01, 0.1, 1  mg/kg PCP (antagonist) -5.7 p.o.), delivered 1 hour prior to T1, increased novel object rec- EP2 (agonist) 4.7 ognition after a 24-hour delay between training and testing to EP4 (agonist) 7.5 a similar degree as the positive control SB399885, delivered 4 IP PGI2 (agonist) -5.1 hours prior to T1 and T2 (F = 11.5, P < .05; Fisher’s PLSD post- (4,54) P2X (agonist) 16.1 hoc test NYX-2925 [0.01, 0.1, 1  mg/kg] or SB399885 vs vehicle, P2Y (agonist) 5.1 P < .05; Figure 5A). 5-HT1A (agonist) 2.5 NYX-2925 (1  mg/kg p.o.) facilitated positive emotional 5-HT1B (antagonist) -6.7 learning and this effect was blocked by pretreatment with 5-HT2A (antagonist) -5.8 a single dose of the NMDAR antagonist CPP as measured by 5-HT2B (agonist) 2.1 both increased rates of hedonic 50-kHz USVs across trials Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 249 Figure 3. NYX-2925 facilitates NMDA current and NMDA receptor-dependent LTP, while decreasing LTD. (A) NYX-2925 (0.1–0.5 µM) enhanced the pharmacologically- isolated NMDAR current in Schaffer collateral-evoked EPSCs in hippocampal CA1 pyramidal neurons. NYX-2925 (B) enhanced the magnitude of LTP at 0.5 µM and (C) decreased the magnitude of LTD at 5 µM of synaptic transmission at Schaffer collateral-CA1 synapses as compared to untreated control (aCSF) slices. (D) NYX-2925 (0.1 and 0.5 µM) enhanced the magnitude of LTP in layer II/III-evoked normalized field EPSP slopes in rat slices recorded in MPFC layer IV. NYX-2925 was bath applied for 40–50 min starting 20 min before high-frequency theta trains to induce LTP (solid bar) or low-frequency stimulation to elicit LTD (solid bar). LTP/LTD was measured 40 min after high- or low-frequency stimulation. Data are shown as mean ± SEM. N = 7 cells per gr oup (A) or 5–10 slices per group (B-D). * P.05, < (A) within-subjects t test vs baseline (B-D) Fishers PLSD post hoc test vs vehicle. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 250 | International Journal of Neuropsychopharmacology, 2018 Figure 4. NYX-2925 persistently enhances hippocampal structural plasticity and metaplasticity following a single in vivo dose (1 mg/kg PO). (A) Plasma and CSF levels following dosing with NYX-2925 (1 mg/kg PO) were measured by LC/MS/MS. The C in the CSF is 44 nM (at 30 min post-dosing) which is well above the threshold for max activing NR2A-D receptors (1 nM) as shown in Figure 2. A single in vivo dose of NYX-2925 (1 mg/kg PO) increased (B) spine head and neck diameter in hippocampal dentate gyrus primary dendrites 24 hrs post-dosing, and (C,D) the magnitude of LTP compared to slices from vehicle-treated control rats prepared (C) 24 hrs and (D) 1 week post-dosing (NYX-2925, 1 mg/kg PO) at Schaffer collateral-CA1 synapses after 3 sub-maximal high-frequency stimulus trains (2 × 100 Hz/800 ms, arrows). Data are shown as Mean ± SEM. N = (A) 2–3 rats, (B) 1412–2034 spines, and (C-D) 6–8 slices per group. (repeated-measures ANOVA: dosing group F = 37.7, P < .05; drug discrimination task (F = 90.9, P < .05; Fisher’s PLSD posthoc (3,31) (6,36) trials F = 39.8, P < .05, dosing group x trials F = 15.0, test ketamine [17 and 30 mg/kg p.o.] vs vehicle, P < .05; Figure  6C). (5,31) (15,31) P < .05, Fisher’s PLSD posthoc test NYX-2925 alone vs all other Unlike ketamine, NYX-2925 did not suppress operant responding groups, P < .05; Figure  5B) and increased running speed to self- at the highest dose tested (F = 9.1, P < .05; Fisher’s PLSD post hoc (6,36) administer heterospecific rough-and-tumble play (F = 12.6, test ketamine [30 mg/kg p.o.] vs vehicle, P < .05; F igure 6D)). (3,31) P < .05; Fisher’s PLSD posthoc test NYX-2925 alone vs all other groups, P < .05; Figure  5C). In addition, NYX-2925 (0.001–1  mg/ Discussion kg p.o.) increased positive emotional learning 1 hour post dos- The NMDA receptor has recently become an attractive target for ing (F = 4.6, P < .05, Fisher’s PLSD posthoc test 0.001, 0.01, 0.1, (5,53) the development of therapeutics for the treatment of a variety of 1 mg/kg vs vehicle, P < .05; data not shown). CNS disorders. Clinical trial data have shown that NMDA receptor modulators have efficacy in mood disorders such as depression, Safety Pharmacology obsessive compulsive disorder, schizophrenia, as well as in neuro- NYX-2925 did no induce sedation/ataxia and does not show pathic pain, among others (Rodriguez et al., 2013 Cain et  ; al., 2014; ketamine-like drug discrimination. NYX-2925 (1–100 mg/kg Feder et al., 2014; Fond et al., 2014; Maher et al., 2017). To date, only p.o.) did not induce a sedative/ataxic effect in the Rota-Rod test rapastinel and DCS have shown efficacy in clinical trials without (F = 0.5, P > .05; Figure 6A) or alter locomotor activity in the the side effects seen with NMDA receptor antagonists. DCS has (3,29) open field test at 1 mg/kg p.o. (F = 0.002, P > .05; Figure 6B). shown a comparatively weak therapeutic response profile and (1,22) Ketamine (p.o.), but not NYX-2925, substituted for a training results have been mixed (Laake and Oeksengaard, 2002; Tuominen dose of ketamine (5.6 mg/kg i.p.) in a dose-dependent manner in the et al., 2005; Ori et al., 2015). Rapastinel is a peptide that must be Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 251 Figure 5. NYX-2925 facilitates learning and memory by activating NMDA receptors. (A) NYX-2925 (0.1–1 mg/kg PO, delivered 1 h prior to training) facilitated novel object recognition as measured by the D2 index [(novel object exploration time – familiar object exploration time) / total exploration time in T2)] tested 24 hrs after training. Pretreatment with a silent dose of the NMDA receptor antagonist CPP blocked the facilitation of learning in the positive emotional learning assay seen with NYX-2925 tested 1 hr after NYX-2925 administration (1 mg/kg PO) as measured by (B) increased rates of hedonic 50-kHz USVs across trial blocks and (C) increased running speed to self-administer heterospecific rough-and-tumble play. Data are shown as mean ± SEM. N = rats 8–12 per group. * P < .05, Fishers PLSD post hoc test (A) vs vehicle or (B) vs all other groups. Figure 6. NYX-2925 does not produce ketamine-like sedative/ataxic or discriminative stimulus effects. (A) NYX-2925 (1, 10, 100 mg/kg PO) did not produce sedation/ ataxia in the fixed speed (16 RPM) RotaRod test. (B) Locomotor activity in the open field was not altered by NYX-2925 (1 mg/kg PO). (C,D) NYX-2925 does not substitute for ketamine as measured by (C) percentage ketamine-lever responding and (D) rates of responding for ketamine (10, 17, 30 mg/kg PO) and NYX-2925 (1, 10, 100 mg/kg PO) in rats trained to discriminate 5.6 mg/kg ketamine from saline. N = 6–12 per group. * Fisher’s PLSD post hoc test vs vehicle. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 252 | International Journal of Neuropsychopharmacology, 2018 administered i.v. To obviate these problems, we developed a pep- D. R. Houck, A. L. Gross, C. Cearley, T. M. Madsen, R. A. Kroes, J. S. tide mimetic program that uses spirocyclic--lactam β chemistry to Burgdorf, and J.  R. Moskal are employees of Aptinyx, Inc., and mimic the dipyrrolidine core of rapastinel. have received financial compensation and stock. The data reported here show that NYX-2925 is an NMDAR modulator that enhances activity-dependent synaptic plasticity in both the hippocampus and MPFC and facilitates learning and Acknowledgments memory. This suggests that NYX-2925, like rapastinel (Moskal The authors thank Puja Kansara, Elizabeth Pollard, Emma et al., 2017), operates through an NMDAR-triggered process that Rodriguez, and Mary Schmidt for their excellent technical leads to lowering of the threshold for induction of LTP that results assistance. in enhancement of learning and memory, as well as long-term metaplasticity that persistently shifts the threshold for induction of LTP. In vivo, NYX-2925 facilitated novel object recognition and References positive emotional learning, both of which are NMDAR-dependent learning tasks (Burgdorf et al., 2011b v; an der Staay et al., 2011), Abraham WC, Bear MF (1996) Metaplasticity: the plasticity of and these effects were blocked by pretreatment with the NMDAR synaptic plasticity. Trends Neurosci 19:126–130. glutamate site antagonist CPP. Further, the dose used to facilitate Alonso E, Lopez-Ortiz F, del Pozo C, Peralta E, Macias A, Gonzalez learning in vivo (1 mg/kg p.o.) led to CSF drug levels that activate J (2001) Spiro beta-lactams as beta-turn mimetics. Design, NR2A-D containing NMDARs in vitro, facilitate both NMDA cur - synthesis, and NMR conformational analysis. J Org Chem rent and NMDAR-dependent LTP in slices from hippocampus and 66:6333–6338. MPFC, and reduce while actually reducing LTD in hippocampus. Bartlett TE, Bannister NJ, Collett VJ, Dargan SL, Massey PV, The observation that NYX-2925 shares with rapastinel an abil- Bortolotto ZA, Fitzjohn SM, Bashir ZI, Collingridge GL, Lodge D ity to simultaneously enhance the induction of LTP, while sup- (2007) Differential roles of NR2A and NR2B-containing NMDA pressing that of LTD (X. L. Zhang et al., 2008), is intriguing, given receptors in LTP and LTD in the CA1 region of two-week old that studies have suggested that LTP and LTD may be preferen- rat hippocampus. Neuropharmacology 52:60–70. tially induced by NMDARs containing NR2B and NR2A subunits, Bittermann H, Gmeiner P (2006) Chirospecific synthesis of spiro- respectively (Bartlett et al., 2007 Morishita et al., ; 2007Y ; ashiro cyclic beta-lactams and their characterization as potent type and Philpot, 2008). This leads us to conclude that NYX-2925, like II beta-turn inducing peptide mimetics. J Org Chem 71:97–102. rapastinel, may selectively activate NR2B-containing NMDARs Bliss TV, Collingridge GL (1993) A synaptic model of mem- (Zhang et al., 2008). In addition, NYX-2925 facilitated learning and ory: long-term potentiation in the hippocampus. Nature memory, metaplasticity of LTP, and structural plasticity, 24 hours 361:31–39. after a single dose, when NYX-2925 was no longer present in the Burgdorf J, Zhang XL, Weiss C, Matthews E, Disterhoft JF, Stanton CSF. Thus, NYX-2925 produces its behavioral effects by facilitat- PK, Moskal JR (2011a) The N-methyl-D-aspartate recep- ing NMDAR-dependent plasticity acutely via direct activation of tor modulator GLYX-13 enhances learning and memory, in NMDAR and chronically by enhancing both metaplasticity and young adult and learning impaired aging rats. Neurobiol structural plasticity triggered by NMDAR activation. Aging 32:698–706. NYX-2925 displays the properties of an attractive therapeutic Burgdorf J, Kroes RA, Weiss C, Oh MM, Disterhoft JF, Brudzynski for NMDAR-modulated CNS disorders. It operates through an SM, Panksepp J, Moskal JR (2011b) Positive emotional learn- NMDAR-triggered, AMPAR-dependent mechanism that leads to ing is regulated in the medial prefrontal cortex by GluN2B- metaplasticity processes similar to LTP, which result in enhance- containing NMDA receptors. Neuroscience 192:515–523. ments in learning and memory as well as long-term metaplasticity Burgdorf J, Zhang XI, Nicholson KL, Balster RL, Leander JD, (Abraham and Bear, 1996). NYX-2925 is orally bioavailable and does Stanton PK, Gross AL, Kroes RA, Moskal JR (2013) GLYX-13, not show ketamine-like side effects or observable adverse effects an NMDA receptor glycine-site functional partial agonist, in toxicology studies, suggesting a wide therapeutic index (>1000). induces antidepressant-like effects without ketamine-like Alonso et  al. (2001) showed that spirocyclic -lactams could β side effects. Neuropsychopharmacology 38:729–742. be synthesized with β -turn conformations making the creation Burgdorf J, Kroes RA, Zhang XL, Gross AL, Schmidt M, Weiss of peptidomimetics using conventional peptide chemistry tech- C, Disterhoft JF, Burch RM, Stanton PK, Moskal JR (2015a) niques possible, and Bittermann and Gmeiner (2006) reported the Rapastinel (GLYX-13) has therapeutic potential for the synthetic methods for β-turn-containing spirocyclic- -lactams β treatment of post-traumatic stress disorder: characteriza- starting from natural proline. Recently, this approach has been tion of a NMDA receptor-mediated metaplasticity process used to develop peptidomimetics of the dopamine receptor mod- in the medial prefrontal cortex of rats. Behav Brain Res ulating peptide L-prolyl-L-leucyl-glycinamide (Khalil et al., 1999) 294:177–185. and the creation of somatostatin mimetics (Lesma et  al., 2013). Burgdorf J, Zhang XL, Weiss C, Gross A, Boikess SR, Kroes RA, Using a similar chemical synthesis approach, we have been able Khan MA, Burch RM, Rex CS, Disterhoft JF, Stanton PK, Moskal to create a novel platform of rapastinel mimetics, exemplified by JR (2015b) The long-lasting antidepressant effects of rapasti- NYX-2925 that may be useful tools to study NMDAR structure and nel (Glyx-13) are associated with a metaplasticity process in function. And since NYX 2925 appears to be an excellent thera- the medial prefrontal cortex and hippocampus. Neuroscience peutic candidate, other compounds from this platform may lead 308:202–211. to additional NMDAR modulators with therapeutic potential. Burgdorf J, Colechio EM, Stanton P, Panksepp J (2017) Positive emotional learning induces resilience to depression: a role for NMDA receptor-mediated synaptic plasticity. Curr Statement of Interest Neuropharmacol 15:3–10. P. K. Stanton is a consultant for Aptinyx, Inc. and has received Cain CK, McCue M, Bello I, Creedon T, Tang DI, Laska E, Goff DC financial compensation and stock. X.-L. Zhang is supported by (2014) d-Cycloserine augmentation of cognitive remediation a grant from Aptinyx, Inc., granted to P. K. Stanton. M. A. Khan, in schizophrenia. Schizophr Res 153:177–183. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 253 Coyle JT (2012) NMDA receptor and schizophrenia: a brief his- Lesma G, Cecchi R, Cagnotto A, Gobbi M, Meneghetti F, Musolino tory. Schizophr Bull 38:920–926. M, Sacchetti A, Silvani A (2013) Tetrahydro-beta-carboline- Cull-Candy S, Brickley S, Farrant M (2001) NMDA receptor subu- based spirocyclic lactam as type II’ beta-turn: application nits: diversity, development and disease. Curr Opin Neurobiol to the synthesis and biological evaluation of somatostatine 11:327–335. mimetics. J Org Chem 78:2600–2610. Danysz W, Parsons CG (1998) Glycine and N-methyl-D-aspartate Lu W, Du J, Goehring A, Gouaux E (2017) Cryo-EM structures of receptors: physiological significance and possible therapeutic the triheteromeric NMDA receptor and its allosteric modula- applications. Pharmacol Rev 50:597–664. tion. Science 355:pii: eaal3729. de Kleine RA, Hendriks GJ, Kusters WJ, Broekman TG, van Maher DP, Chen L, Mao J (2017) Intravenous ketamine infusions Minnen A (2012) A randomized placebo-controlled trial of for neuropathic pain management: a promising therapy in D-cycloserine to enhance exposure therapy for posttrau- need of optimization. Anesth Analg 124:661–674. matic stress disorder. Biol Psychiatry 71:962–968. Matsuzaki M, Honkura N, Ellis-Davies GC, Kasai H (2004) Dolino DM, Cooper D, Ramaswamy S, Jaurich H, Landes CF, Structural basis of long-term potentiation in single dendritic Jayaraman V (2015) Structural dynamics of the glycine- spines. Nature 429:761–766. binding domain of the N-methyl-D-aspartate receptor. J Biol Millecamps M, Centeno MV, Berra HH, Rudick CN, Lavarello S, Chem 290:797–804. Tkatch T, Apkarian AV (2007) D-cycloserine reduces neuro- Feder A, Parides MK, Murrough JW, Perez AM, Morgan JE, Saxena pathic pain behavior through limbic NMDA-mediated cir - S, Kirkwood K, Aan Het Rot M, Lapidus KA, Wan LB, Iosifescu cuitry. Pain 132:108–123. D, Charney DS (2014) Efficacy of intravenous ketamine for Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC treatment of chronic posttraumatic stress disorder: a rand- (2007) Activation of NR2B-containing NMDA receptors is not omized clinical trial. JAMA Psychiatry 71:681–688. required for NMDA receptor-dependent long-term depres- Fond G, Loundou A, Rabu C, Macgregor A, Lancon C, Brittner M, sion. Neuropharmacology 52:71–76. Micoulaud-Franchi JA, Richieri R, Courtet P, Abbar M, Roger Morris RG (2013) NMDA receptors and memory encoding. M, Leboyer M, Boyer L (2014) Ketamine administration in Neuropharmacology 74:32–40. depressive disorders: a systematic review and meta-analysis. Moskal JR, Burgdorf JS, Stanton PK, Kroes RA, Disterhoft JF, Burch Psychopharmacology (Berl) 231:3663–3676. RM, Khan MA (2017) The development of rapastinel (formerly Ghasemi M, Phillips C, Trillo L, De Miguel Z, Das D, Salehi A (2014) GLYX-13); a rapid acting and long lasting antidepressant. Curr The role of NMDA receptors in the pathophysiology and treat- Neuropharmacol 15:47–56. ment of mood disorders. Neurosci Biobehav Rev 47:336–358. Moskal JR, Kuo AG, Weiss C, Wood PL, O’Connor Hanson A, Kelso Ghasemi M, Schachter SC (2011) The NMDA receptor complex S, Harris RB, Disterhoft JF (2005) GLYX-13: a monoclonal anti- as a therapeutic target in epilepsy: a review. Epilepsy Behav body-derived peptide that acts as an N-methyl-D-aspartate 22:617–640. receptor modulator. Neuropharmacology 49:1077–1087. Goff DC (2012) D-cycloserine: an evolving role in learning and Nadeson R, Tucker A, Bajunaki E, Goodchild CS (2002) Potentiation neuroplasticity in schizophrenia. Schizophr Bull 38:936–941. by ketamine of fentanyl antinociception. I. An experimental Haring R, Stanton PK, Scheideler MA, Moskal JR (1991) Glycine- study in rats showing that ketamine administered by non- like modulation of N-methyl-D-aspartate receptors by a spinal routes targets spinal cord antinociceptive systems. Br monoclonal antibody that enhances long-term potentiation. J Anaesth 88:685–691. J Neurochem 57:323–332. Noguchi J, Matsuzaki M, Ellis-Davies GC, Kasai H (2005) Spine- Harris RE, Sundgren PC, Pang Y, Hsu M, Petrou M, Kim SH, neck geometry determines NMDA receptor-dependent Ca2+ McLean SA, Gracely RH, Clauw DJ (2008) Dynamic levels of signaling in dendrites. Neuron 46:609–622. glutamate within the insula are associated with improve- Ori R, Amos T, Bergman H, Soares-Weiser K, Ipser JC, Stein DJ ments in multiple pain domains in fibromyalgia. Arthritis (2015) Augmentation of cognitive and behavioural therapies Rheum 58:903–907. (CBT) with d-cycloserine for anxiety and related disorders. Hirst WD, Stean TO, Rogers DC, Sunter D, Pugh P, Moss SF, Cochrane Database Syst Rev:CD007803. Bromidge SM, Riley G, Smith DR, Bartlett S, Heidbreder CA, Ota KT, Liu RJ, Voleti B, Maldonado-Aviles JG, Duric V, Iwata M, Atkins AR, Lacroix LP, Dawson LA, Foley AG, Regan CM, Upton Dutheil S, Duman C, Boikess S, Lewis DA, Stockmeier CA, N (2006) SB-399885 is a potent, selective 5-HT6 receptor antag- DiLeone RJ, Rex C, Aghajanian GK, Duman RS (2014) REDD1 onist with cognitive enhancing properties in aged rat water is essential for stress-induced synaptic loss and depressive maze and novel object recognition models. Eur J Pharmacol behavior. Nat Med 20:531–535. 553:109–119. Paoletti P, Bellone C, Zhou Q (2013) NMDA receptor subunit div -er Iacobucci GJ, Popescu GK (2017) NMDA receptors: linking physio- sity: impact on receptor properties, synaptic plasticity and logical output to biophysical operation. Nat Rev Neurosci disease. Nat Rev Neurosci 14:383–400. 18:236–249. Parent MA, Wang L, Su J, Netoff T, Yuan LL (2010) Identification of Ishiyama S, Brecht M (2016) Neural correlates of ticklishness in the hippocampal input to medial prefrontal cortex in vitro. the rat somatosensory cortex. Science 354:757–760. Cereb Cortex 20:393–403. Karakas E, Furukawa H (2014) Crystal structure of a heterotetra- Patrizi A, Picard N, Simon AJ, Gunner G, Centofante E, Andrews NA, meric NMDA receptor ion channel. Science 344:992–997. Fagiolini M (2016) Chronic administration of the N-methyl- Khalil EM, Ojala WH, Pradhan A, Nair VD, Gleason WB, Mishra D-aspartate receptor antagonist ketamine improves Rett RK, Johnson RL (1999) Design, synthesis, and dopamine Syndrome phenotype. Biol Psychiatry 79:755–764. receptor modulating activity of spiro bicyclic peptid- Preskorn S, Macaluso M, Mehra DO, Zammit G, Moskal JR, Burch omimetics of L-prolyl-L-leucyl-glycinamide. J Med Chem RM, Group GCS (2015) Randomized proof of concept trial of 42:628–637. GLYX-13, an N-methyl-D-aspartate receptor glycine site par - Laake K, Oeksengaard AR (2002) D-cycloserine for Alzheimer’s tial agonist, in major depressive disorder nonresponsive to a disease. Cochrane Database Syst Rev:CD003153. previous antidepressant agent. J Psychiatr Pract 21:140–149. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 254 | International Journal of Neuropsychopharmacology, 2018 Pyke T, Osmotherly PG, Baines S (2016) Measuring glutamate lev- van der Staay FJ, Rutten K, Erb C, Blokland A (2011) Effects of the els in the brains of fibromyalgia patients and a potential role cognition impairer MK-801 on learning and memory in mice for glutamate in the pathophysiology of fibromyalgia symp- and rats. Behav Brain Res 220:215–229. toms: a systematic review. Clin J Pain 33:944–954. Vasilescu AN, Schweinfurth N, Borgwardt S, Gass P, Lang Robb CM (1991) Restrictive covenant law in Georgia: back to the UE, Inta D, Eckart S (2017) Modulation of the activity of drawing board. J Med Assoc Ga 80:546–548. N-methyl-d-aspartate receptors as a novel treatment Rodriguez CI, Kegeles LS, Levinson A, Feng T, Marcus SM, option for depression: current clinical evidence and thera- Vermes D, Flood P, Simpson HB (2013) Randomized con- peutic potential of rapastinel (GLYX-13). Neuropsychiatr Dis trolled crossover trial of ketamine in obsessive-compulsive Treat 13:973–980. disorder: proof-of-concept. Neuropsychopharmacology 38: Wilkinson D, Wirth Y, Goebel C (2014) Memantine in patients 2475–2483. with moderate to severe Alzheimer’s disease: meta-analyses Rodriguez CI, Zwerling J, Kalanthroff E, Shen H, Filippou M, Jo using realistic definitions of response. Dement Geriatr Cogn B, Simpson HB, Burch RM, Moskal JR (2016) Effect of a novel Disord 37:71–85. NMDA receptor modulator, rapastinel (formerly GLYX-13), in Yashiro K, Philpot BD (2008) Regulation of NMDA receptor sub- OCD: proof of concept. Am J Psychiatry 173:1239–1241. unit expression and its implications for LTD, LTP, and meta- Tavoloni N, Schaffner F (1989) Bile secretory apparatus in the plasticity. Neuropharmacology 55:1081–1094. newborn dog: relationship between structural and functional Zhang L, Xu T, Wang S, Yu L, Liu D, Zhan R, Yu SY (2013) NMDA immaturities. Biol Neonate 55:124–135. GluN2B receptors involved in the antidepressant effects of cur - Thompson LT, Moskal JR, Disterhoft JF (1992) Hippocampus- cumin in the forced swim test. Prog Neuropsychopharmacol dependent learning facilitated by a monoclonal antibody or Biol Psychiatry 40:12–17. D-cycloserine. Nature 359:638–641. Zhang XL, Sullivan JA, Moskal JR, Stanton PK (2008) A NMDA Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, receptor glycine site partial agonist, GLYX-13, simultan- Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010) eously enhances LTP and reduces LTD at Schaffer collat- Glutamate receptor ion channels: structure, regulation, and eral-CA1 synapses in hippocampus. Neuropharmacology function. Pharmacol Rev 62:405–496. 55:1238–1250. Tuominen HJ, Tiihonen J, Wahlbeck K (2005) Glutamatergic drugs Zhou HY, Chen SR, Pan HL (2011) Targeting N-methyl-D-aspartate for schizophrenia: a systematic review and meta-analysis. receptors for treatment of neuropathic pain. Expert Rev Clin Schizophr Res 72:225–234. Pharmacol 4:379–388. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Neuropsychopharmacology Oxford University Press

NYX-2925 Is a Novel NMDA Receptor-Specific Spirocyclic-β-Lactam That Modulates Synaptic Plasticity Processes Associated with Learning and Memory

Free
13 pages

Loading next page...
 
/lp/ou_press/nyx-2925-is-a-novel-nmda-receptor-specific-spirocyclic-lactam-that-u4rqx6NVVT
Publisher
Oxford University Press
Copyright
© The Author(s) 2017. Published by Oxford University Press on behalf of CINP.
ISSN
1461-1457
eISSN
1469-5111
D.O.I.
10.1093/ijnp/pyx096
Publisher site
See Article on Publisher Site

Abstract

Background: N-methyl-D-aspartate receptors are one member of a family of ionotropic glutamate receptors that play a pivotal role in synaptic plasticity processes associated with learning and have become attractive therapeutic targets for diseases such as depression, anxiety, schizophrenia, and neuropathic pain. NYX-2925 ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo- 2,5-diazaspiro[3.4]octan-2-yl)butanamide) is one member of a spiro-β-lactam-based chemical platform that mimics some of the dipyrrolidine structural features of rapastinel (formerly GLYX-13: threonine-proline-proline-threonine) and is distinct from known N-methyl-D-aspartate receptor agonists or antagonists such as D-cycloserine, ketamine, MK-801, kynurenic acid, or ifenprodil. Methods: The in vitro and in vivo pharmacological properties of NYX-2925 were examined. Results: NYX-2925 has a low potential for “off-target” activity, as it did not exhibit any significant affinity for a large panel of neuroactive receptors, including hERG receptors. NYX-2925 increased MK-801 binding to human N-methyl-D-aspartate receptor NR2A-D subtypes expressed in HEK cells and enhanced N-methyl-D-aspartate receptor current and long-term potentiation (LTP) in rat hippocampal slices (100–500 nM). Single dose ex vivo studies showed increased metaplasticity in a hippocampal LTP paradigm and structural plasticity 24 hours after administration (1 mg/kg p.o.). Significant learning enhancement in both novel object recognition and positive emotional learning paradigms were observed (0.01–1 mg/kg p.o.), and these effects were blocked by the N-methyl-D-aspartate receptor antagonist CPP. NYX-2925 does not show any addictive or sedative/ataxic side effects and has a therapeutic index of >1000. NYX-2925 (1 mg/kg p.o.) has a cerebrospinal fluid half-life of 1.2 hours with a Cmax of 44 nM at 1 hour. Conclusions: NYX-2925, like rapastinel, activates an NMDA receptor-mediated synaptic plasticity process and may have therapeutic potential for a variety of NMDA receptor-mediated central nervous system disorders. Keywords: NMDA receptor, learning and memory, synaptic plasticity Received: August 2, 2017; Revised: October 7, 2017; Accepted: October 17, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any 242 medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 243 Significance Statement NYX-2925 is a novel NMDA receptor-specific modulator that facilitates synaptic plasticity and has therapeutic potential for a v - ar iety of NMDA receptor-mediated central nervous system (CNS) disorders. NYX-2925 was synthesized using a novel spirocyclic-β- lactam chemical approach using rapastinel (formerly GLYX-13) as a template, which was in turn synthesized from a hypervariable region of a unique monoclonal antibody with NMDA receptor modulatory properties. Thus, the creation of NYX-2925 completes the process of developing monoclonal antibodies to help elucidate the molecular mechanisms of complex biological processes such as learning and memory and converting them into small molecules with therapeutic potential. Introduction N-methyl-D-aspartate (NMDA) receptors are one of a family of and activity profiles. NYX-2925 is a representative compound ligand gated ionotropic glutamate receptors that are found pre- from this platform. Figure  1 shows the structure of rapastinel dominantly in the CNS and developmentally regulated (Cull- and NYX-2925, and we report on its pharmacological, toxico- Candy et al., 2001; Traynelis et al., 2010). They are unique among logical, functional, and mechanistic properties. glutamate receptors in that they require both glutamate and glycine for full activation (Danysz and Parsons, 1998). They are Materials and Methods heterotetrameric complexes that are expressed as multiple sub- types each with unique properties (Paoletti et al., 2013). NMDA Animals receptors play a pivotal role in modulating normal neuronal functions including activity-dependent synaptic plasticity asso- Adult male Sprague-Dawley rats from Harlan or Charles River ciated with learning and memory (Bliss and Collingridge, 1993; were used for most studies. For the novel object recognition Yashiro and Philpot, 2008; Morris, 2013) and have been impli- study, adult male Lister Hooded rats from Harlan were used. cated in a variety of CNS disorders, including schizophrenia Rats were group housed (3–4 per cage) in Lucite cages with aspen (Coyle, 2012; Goff, 2012), mood disorders (Ghasemi et  al., 2014; wood chip bedding, maintained on a 12-hour-light/12-hour-dark Vasilescu et  al., 2017), epilepsy (Ghasemi and Schachter, 2011), cycle (lights on at 5:00 am), and given ad libitum access to Purina neuropathic pain (Millecamps et  al., 2007Zhou ; et  al., 2011), Lab Chow and tap water throughout the study. For the drug dis- fibromyalgia (Harris et al., 2008; Pyke et al., 2016), Rett syndrome crimination study, rats were singly housed with ad libitum access (Patrizi et  al., 2016), and cognitive decline due to normal aging to water. All experiments were approved by the Northwestern (Robb, 1991; Burgdorf et al., 2011a) among others. University, Virginia Commonwealth University, and New York Recently, reports have described positive clinical trial Medical College Institutional Animal Care and Use Committees. data with mechanistically distinct NMDA receptor modula- tors including rapastinel (formerly GLYX-13) and ketamine for Drugs depression (Fond et al., 2014; Preskorn et al., 2015) and obsessive- compulsive disorder (Rodriguez et al., 20132016 , ), D-cycloserine NYX-2925 was synthesized by Sai Life Sciences (India) and was for schizophrenia (Cain et  al., 2014) and posttraumatic stress administered p.o. (0.1–10  mg/kg) in 1  mL/kg in 0.5% carboxy- disorder (de Kleine et al., 2012), and memantine for Alzheimer’s methylcellulose (CMC) 0.9% sterile saline. The NMDA receptor disease (Wilkinson et al., 2014). The key role that NMDA recep- glutamate site antagonist CPP ((±)-3-(2-carboxypiperazin-4-yl) tors play in synaptic plasticity throughout the CNS, the marked propyl-1-phosphonic acid) was purchased from Sigma and increase in NMDA receptor mechanistic studies, including X-ray administered i.p. (10  mg/kg) in 1  mL/kg 0.9% sterile saline. The crystallographic analysis (Karakas and Furukawa, 2014 Dolino ; dose of CPP (10 mg/kg i.p.) was chosen based on previous reports et  al., 2015; Lu et  al., 2017), and biophysical studies on recep- that this dose could block the antidepressant-like effects of an tor subtype properties (Tavoloni and Schaffner, 1989 Iacobucci ; NMDAR positive modulator without exhibiting behavioral effects and Popescu, 2017), coupled with clinical trial successes seen on its own (L. Zhang et  al., 2013; Burgdorf et  al., 2015b). The with NMDA receptor modulators, make this receptor complex 5-HT receptor antagonist SB399885 was purchased from GVK an attractive target for drug discovery. Biosciences (India) and was administered p.o. (10 mg/kg) in 2 mL/ Rapastinel is a tetrapeptide (threonine-proline-proline- kg 1% CMC and was used as a positive control in the novel object threonine) derived from a hypervariable region of a monoclonal recognition study (Hirst et al., 2006). Ketamine-HCl (Ketalar) was antibody B6B21 (Moskal et al., 2005). B6B21 was shown to act as obtained from Patterson Veterinary Inc. and was diluted with a cognitive enhancer (Thompson et  al., 1992) with glycine-site 0.9 % saline to the concentration required to provide the desired partial agonist properties at the NMDA receptor (Haring et  al., dose in a 1-mL/kg volume for both i.p. and p.o. administration. 1991). Rapastinel has also been found to be a robust cognitive enhancer with marked antidepressant-like effects in a variety of [ H] MK-801 Potentiation Assay rat models (Burgdorf et al., 2013 2017 , ). Mechanistically, rapasti- nel appears to bind directly to NMDA receptors, triggering an NMDAR Subtype Expressing HEK Cell Membrane Preparation increase in AMPA receptor activity and leading to a long-term Crude membranes were prepared using transiently transfected, potentiation-like increase in synaptic plasticity associated with NMDAR-expressing HEK cells, described below. All procedures learning (Moskal et al., 2017). were performed at 4°C. Briefly, pelleted cells were initially A key structural feature of rapastinel is its dipyrrolidine- washed in 10 mM Tris acetate (pH 7.4 at 4°C), pelleted, and fro- based β-turn motif. A novel chemical platform was created using zen at -80°C overnight. The pellet was then resuspended and spirocyclic-β-lactam chemistry (Bittermann and Gmeiner, 2006) homogenized (30 strokes) in a glass homogenizer and pelleted with a variety of NMDA receptor subtype selectivity, potency, at 51500 x g for 30 minutes at 4°C and stored at –80ºC until assay. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 244 | International Journal of Neuropsychopharmacology, 2018 Figure  1. Structural comparison of the peptide rapastinel with the spirocyclic β-lactam, ((2S, 3R)-3-hydroxy-2-((R)-5-isobutyryl-1-oxo-2,5-diazaspiro[3.4]octan-2-yl) butanamide) (NYX-2925). Spirocyclic β-lactam chemistry has been used to create a number of new molecules that mimic key properties of their parent peptides (Bittermann and Gmeiner, 2006). Functional glycine site agonist effects were measured using Bioavailability an [ H] MK-801 potentiation assay. Briefly, 300 µg of membrane Male Sprague Dawley rats were dosed with NYX-2925 (1 mg/kg extract protein were preincubated for 15 minutes at 25°C in the p.o.), and jugular vein blood draws taken in K2-EDTA-treated presence of a saturating concentration of glutamate (50  µM) and tubes and cisterna magna cerebrospinal fluid (CSF) draws were varying concentrations of NYX-2925. Following the addition of taken at various time points. Samples were maintained at 4°C 0.3mCi [ H] MK-801 (Amersham, 22.5 Ci/mmol), reactions were for 30 to 60 minutes after collection and stored at 80°C until incubated for an additional 15 minutes (nonequilibrium con- assay. On the day of the assay, plasma, CSF, and standards were ditions). Bound and free [H] MK-801 were separated via rapid thawed at 4°C. Samples were extracted with acetonitrile and filtration. Zero levels were determined in the absence of any NYX-2925 levels were assessed by liquid chromatography tan- glycine ligand. The percent maximal [H] MK-801 binding was dem mass spectrometry and the lower limit of quantification for calculated relative to stimulation measured in the presence of this assay was ~4 nM. 1 mM glycine and 50 µM glutamate. Binding curves were fitted using GraphPad software. Dendritic Spine Morphology Analysis Creation of the Stable hNMDAR1-Expressing HEK Dendritic spine analyses were conducted as previously described Cell Line (Ota et  al., 2014; Burgdorf et  al., 2015b) using the Afraxis ESP The cDNA encoding the human GluN1-1 (GenBank BC156961) Platform (Afraxis, Inc.). Animals were given a single dose of was amplified from MGC clone 100063609 using pfu polymerase NYX-2925 (1 mg/kg p.o.), or 0.5% Na-CMC in 0.9% sterile saline and subcloned into the pCMV/zeoDNA3 vector using standard vehicle (1  mL/kg), and 24 hours post dosing they were deeply molecular techniques and verified by direct sequencing. HEK anesthetized (isoflurane) and brains fixed via cardiac perfusion cells (ATCC) were transfected with the mutant construct using using 4% paraformaldehyde. Brains were stored in ice cold 0.1 X-tremeGENE 9 transfection reagent (Roche) and stable clones M phosphate buffer and stored at 4°C until sectioning. Brains selected in Zeocin-containing media. were sectioned using a Vibratome (Leica VT1000) to collect sec- tions (300 μm thick) from the anterior to posterior extremes of each brain. Ballistic dye labeling (DiI and DiO; 3  mg dissolved Creation of the Transient hNMDAR2 (A-D) in methylene chloride and coated on tungsten particles) was Expression Vectors performed using a commercially available gene gun (Bio-Rad) cDNAs encoding the hNMDAR2A (GENBANK #NM_000833, to label neurons. Thick sections were mounted to slides with pfu polymerase amplified from human cortex cDNA), hNM- raised barriers using ProLong Gold (Life Technologies) and cover DAR2B (GENBANK #NM_000834, pfu polymerase amplified slipped. Laser-scanning confocal microscopy (Olympus FV1000) from Open Systems clone #8322670), hNMDAR2C (GENBANK was performed using a 63 × objective (1.42 NA) to scan individu- #NM_000835, pfu polymerase amplified from OriGene clone ally labeled neurons at high resolution (0.103 × 0.103 × 0.33  μm #SC300138), and hNMDAR2D (GENBANK #NM_000836, pfu voxels). Microscopy was performed blind to experimental condi- polymerase amplified from OriGene clone #SC300139) were tions. A minimum of 5 cells per animal were sampled. Primary subcloned into the pCMV6/XL5 vector using standard mo- dendrites within the inner molecular layer were analyzed, and lecular techniques. samples (50 μm) were collected from primary dendrites starting at 100 μm from the soma. Spine head and neck sizes were ana- lyzed given that larger spine head and necks are associated with Off-Target Receptor Binding Assays greater NMDAR-mediated calcium flux (Noguchi et al., 2005) as To further characterize the target specificity of NYX-2925, we well as the induction of LTP (Matsuzaki et al., 2004). tested the ability of 10  μM NYX-2925 (>10 000 × Cmax) to com- Blind deconvolution (AutoQuant) was applied to raw 3‐ pete for binding in a radioligand displacement assay using a dimensional digital images that were then analyzed for spine broad panel of known CNS protein targets (LeadProfilingScreen density and morphology by trained analysts. Individual spines 2, Eurofins Cerep). The binding panel included adenosine were measured manually for (a) head diameter, (b) spine and adrenergic receptor sites; dopamine, histamine, and opi- length, and (c) spine neck diameter from image Z-stacks using oid receptors; calcium channels; and muscarinic cholinergic software custom-designed by Afraxis Inc. Each dendrite was receptor sites. analyzed by 3 to 4 independent analysts. Analysts were blinded Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 245 to all experimental conditions (including treatment, brain stimulation induction. NYX-2925 was bath applied for 40 to region, and cell type). 50 minutes starting 20 minutes before application of high-fre- quency stimulation. Extracellular Hippocampal Recordings Metaplasticity Studies Experiments were conducted as described previously (Burgdorf Experiments were conducted as described previously (Zhang et  al., 2015b). Rats were dosed with NYX-2925 (1  mg/kg p.o.) et al., 2008; Burgdorf et al., 2015b). Adult male rats were deeply and hippocampal slices were prepared either 24 hours or 1 anesthetized with isoflurane and decapitated. Brains were week post dosing. Three submaximal bouts of high-frequency removed rapidly, submerged in ice-cold artificial CSF (ACSF, Schaffer collateral stimulation (2 × 100 Hz/800  ms) were applied 2–4°C), which contained (in mM): 124 NaCl, 4 KCl, 1.5 MgCl, 2.5 20 minutes apart. LTP was measured 40 minutes after the last CaCl , 1.25 NaH PO , 26 NaHCO , 10 glucose; at pH 7.4, gassed 2 2 4 3 high-frequency bout of stimulation. continuously with 95% O/5% CO . Brains were hemisected, the 2 2 frontal lobes removed, and individual hemispheres glued using cyanoacrylate adhesive onto a stage immersed in ice-cold ACSF Intracellular Recordings from Hippocampal CA1 gassed continuously with 95% O/5% CO during slicing. Then 2 2 Pyramidal Neurons 400-μm-thick coronal slices were cut using a Vibratome (Leica Whole-cell patch clamp recordings from CA1 pyramidal neurons VT1200S) and transferred to an interface holding chamber for were acquired as described previously (Burgdorf et  al., 2013). incubation at room temperature for a minimum of 1 hour be- Patch pipette resistance was 6 to 6.5 MΩ when filled with intra- fore transferring to a Haas-style interface recording chamber cellular solution that contained (in mM): 135 CsMeSO , 8 NaCl, continuously perfused at 3  mL/min with oxygenated ACSF at 10 HEPES, 0.2 EGTA, 2 Mg-ATP, 0.3 Na-GTP, and 1 QX-314 [N-(2,6- 32 ± 0.5°C. dimethylphenylcarbamoylthyl)-triethylammonium bromide], Low resistance recording electrodes were made from thin- 275 mOsm, pH 7.25 adjusted with Cs(OH) . CA1 pyramidal neu- walled borosilicate glass (1–2 MΩ after filling with ACSF) and rons were visualized by infrared imaging and patched using a inserted into the apical dendritic region of the Schaffer collat- 60x water-immersed objective mounted to a Zeiss microscope eral termination field in stratum radiatum of the CA1 region (Axioskop 2 Fs plus). After whole-cell voltage clamp configura- to record field excitatory postsynaptic potentials (fEPSPs). A bi- tion was established, access resistance was carefully monitored, polar stainless-steel stimulating electrode (FHC) was placed on and only cells with stable access resistance (<5% change) were Schaffer collateral-commissural fibers in CA3 stratum radiatum, included in analyses. Excitatory postsynaptic currents (EPSCs) and constant current stimulus intensity adjusted to evoke ap- were recorded using a MultiClamp 700B (Molecular Devices); proximately half-maximal fEPSPs once each 30 seconds (50–100 with the low-pass filter setting at 1 to 3 kHz, series resistance pA; 100-μs duration). fEPSP slope was measured before and after was compensated in the voltage-clamp mode, and patched cells induction of LTP or LTD by linear interpolation from 20% to 80% whose series resistance changed by >10% were rejected from of maximum negative deflection, and slopes confirmed to be analysis. Signals were filtered at 3 kHz and digitized at 10 kHz stable to within ± 10% for at least 15 minutes before commencing with a Digidata 1322A controlled by a Clampex (v9.2) (Molecular an experiment. Signals were recorded using a Multiclamp 700B Devices). A  bipolar tungsten stimulating electrode (FHC) was amplifier and digitized with a Digidata 1322 (Axon Instruments). placed in the Schaffer collateral-commissural fibers in CA3 Data were analyzed using pClamp software (version 9, Axon stratum radiatum and stimulus pulses (800  µS duration) were Instruments). delivered at 15- to 30-second intervals. Neurons were voltage clamped at -70 mV to record EPSCs to assess input-output rela- Hippocampal LTP and LTD Studies tions and paired-pulse facilitation. Neurons were clamped at -40 Experiments were conducted as described previously (Zhang mV for recording NMDA currents to relieve voltage-dependent et al., 2008). LTP was induced by 2 theta trains pared by 3 min- magnesium block, and slices were perfused with ACSF contain- utes (3× 100 Hz/500  ms) and LTD induced by a low-frequency ing 0 added magnesium, 3  mM calcium, 10  µM picrotoxin, and stimulus train (2 Hz/10 min). LTP/LTD was measured 40 minutes 10 µM CNQX to isolate NMDA conductances. after high- or low-frequency stimulation. NYX-2925 was bath All recording pipette solutions were made with deionized applied for 40 to 50 minutes starting 20 minutes before applica- −2 distilled water (resistance >18 M Ω cm ; Milli-Q system). Data tion of high- or low-frequency stimulation. were analyzed initially with Clampfit (v9) (Axon Instruments) and further processed and presented with Origin 6.1 (Microcal Medial Prefrontal Cortex (MPFC) LTP Studies Software) and CorelDraw 10.0 (Corel) programs. Experiments were conducted as described previously (Burgdorf et  al., 2015a). Four-hundred-μm thick slices were cut using a vibratome (Leica VT1200S) in a modified coronal orientation Novel Object Recognition (NOR) (Parent et  al., 2010) containing both prelimbic and infralimbic regions of the MPFC that are targets (via the fornix) of the hip- Experiments were conducted as described previously (Hirst et  al., 2006). Rats were habituated to the NOR test box twice a pocampal-MPFC pathway. Recording electrodes were inserted into layer III/IV of the prelimbic MPFC and monosynaptically day for 2 consecutive days prior to testing. Each habituation ses- sion was comprised of a 3-minute exposure to the empty test evoked fEPSPs evoked by mixed excitatory inputs were recorded from layer V pyramidal neurons. A bipolar tungsten stimulating box (46 x 30 x 45  cm), followed by 1 minute in the side annex (1 x 30 x 45 cm) and a further 3 minutes in the test box, thereby electrode (FHC) was placed on MPFC deep white matter. LTP was induced by stimulation of input axons with 3 high-frequency mimicking the test protocol. A  vehicle dose was administered prior to one of the habituation sessions on each habituation day. theta burst stimulus trains of 10  ×  100 Hz/5 pulse bursts each, applied at an inter-burst interval of 200 milliseconds. Each train The NOR test comprised 2 test sessions, T1 and T2, each last- ing 3 minutes. On the first test day, T1, rats were habituated to was 2 seconds in duration, and trains were applied 3 minutes apart. LTP/LTD was measured 40 minutes after high-frequency the empty test box for 3 minutes and then placed in the side Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 246 | International Journal of Neuropsychopharmacology, 2018 annex for approximately 1 minute whilst 2 identical test objects minutes using a within-subjects design. Habituation and test- were placed in the test arena equally spaced to each other and ing consisted of placing rats onto the fixed speed version of the the side walls. The rat was then returned to the test arena and Rota-Rod test (16 RPM) for 300 seconds, and the latency to fall off allowed to freely explore the objects for a further 3 minutes. At the Rota-Rod was recorded. the end of the test session, the rat was returned to its home cage. Following a 24-hour inter-trial interval, the recall trial (T2) Open Field Test of Locomotor Activity was conducted. T2 was similar to T1 except that one of the “fa- Open field testing was conducted as previously described miliar” objects was substituted for a novel one of a similar size (Burgdorf et al., 2013). Rats were placed into the center chamber of and color but different shape. The objects used were made of the open field (40 x 40 x 20 cm) for 10 minutes under dim-red light- black hardened plastic and were geometric shapes (towers and ing 1 hour post dosing with NYX-2925 (1  mg/kg p.o.) or vehicle pyramids) that were of no relevance to the animals. Objects were (1 mL/kg p.o.). Line crosses were scored offline from video record- cleaned between trials with 70% ethanol to eliminate odor cues. ings by an experimenter blinded to the treatment condition. T1 and T2 trials were recorded by GeoVision surveillance camera software and files were saved on DVD for remote scor- ing by an operator blinded to the treatments. Exploration was Drug Discrimination scored as time spent sniffing or licking the objects, when the Testing was conducted as described previously (Burgdorf nose was in contact with the object and was moving (i.e., when et  al., 2013). Adult male Sprague-Dawley rats were trained to the animal was sniffing). Sitting on the object or next to it with discriminate ketamine (5.6 mg/kg, i.p.) from saline (1 mL/kg, i.p.) the nose directed away was not classed as exploration. The dis- 5  d/wk (Monday-Friday; 15  min/d) in standard 2-lever operant crimination index [(time spent exploring the novel object - time conditioning chambers (Med Associates) under a double-alter - spent exploring the familiar object)/total exploration time] was nation schedule of ketamine or saline (K, K, S, S, K, K, etc.). Rats then calculated for T2. were placed in the operant conditioning chamber 10 minutes For the NYX-2925 groups, 0.01, 0.1, or 1  mg/kg p.o. was post-injection and the session initiated as signaled by illumina- administered 60 minutes before T1 and CMC saline adminis- tion of the chamber house light. Completion of 10 consecutive tered 1 hour before T2. For the SB399885 group, 10  mg/kg p.o. responses (fixed ratio 10; FR 10) on the correct lever resulted in was administered 240 minutes before both T1 and T2. For the ve- delivery of a 45-mg food pellet (Bio-Serv) and illumination of a hicle group, CMC saline (1 mL/kg) was administered 60 minutes white stimulus light over the lever. Incorrect responding reset before both T1 and T2. the FR for correct-lever responding. Food (Harlan Teklad Rodent Diet) access beyond those obtained during behavioral ses- Ultrasonic Vocalization (USV) Assay sions was restricted to ~20 g given post session. Animals were tested following administration of various doses of NYX-2925 Heterospecific rough-and-tumble play was conducted as pre- (1, 10, 100 mg/kg, p.o.), CMC saline (2 mL/kg) or ketamine (10, 17 viously described (Burgdorf et  al., 2011b). The experimenter 30 mg/kg, p.o.), and lever selection and rates of responding were was blind to the treatment condition of the animals. Animals recorded. Doses of NYX-2925 and its vehicle were administered received 3 minutes of heterospecific rough-and-tumble play 1 hour prior to the session start and p.o. ketamine was adminis- consisting of alternating 15-second blocks of heterospecific tered 15 minutes prior to session start. For all p.o. test sessions, play and 15 seconds of no stimulation. High-frequency USVs rats were also administered i.p. saline, 1 mL/kg, 10 minutes prior were recorded and analyzed by sonogram in a blind manner to session start to mimic training conditions. Training contin- as described previously (Burgdorf et  al., 2011b). At the end of ued under the double alternation of 5.6  mg/kg ketamine and the 3-minute session, the latency for the rat to approach the saline injections between test sessions. Illumination of lights, experimenters hand to self-administer heterospecific play was recording of responses, and pellet delivery were performed with also measured. Animals were not habituated to play stimula- MED-PC operant conditioning software (version 1.1). tion before dosing and testing. Using this paradigm, the in- crease in 50-kHz USVs that occurs across trial blocks reflects positive emotional learning (Burgdorf et  al., 2011b Ishiy ; ama Statistical Analysis and Brecht, 2016). Behavioral and electrophysiological data were analyzed by Animals were administered NYX-2925 (1 mg/kg p.o.) or CMC ANOVA, followed by Fisher’s PLSD posthoc test (Statview). The saline (p.o.) 1 hour before testing. Rats received the NMDA re- level of statistical significance was set at P < .05. ceptor glutamate site antagonist CPP (10  mg/kg i.p.) or sterile Data from the [H] MK-801 potentiation assay data was ana- saline (i.p.) 2 hours before NYX-2925 (1 mg/kg p.o.) or CMC sa- lyzed by Prism (Graphpad). line (p.o.). This dose of NYX-2925 was chosen given that it leads to CSF exposures that activate all 4 NMDAR subtypes (Figures 2A-D and 4A) and showed maximal facilitation of learning and Results memory in the novel object recognition test (Figure 5A). [ H] MK-801 Potentiation and Off-target Receptor Rota-Rod Test of Motor Coordination Binding Rota-Rod testing was conducted as previously described NYX-2925 facilitated [H] MK-801 binding in all four human (Nadeson et al., 2002) using a 4-station Rota-Rod apparatus (Med NMDAR2 subtypes. The concentration-response curves for NYX- Associates). One day before testing, animals received 3 Rota-Rod 2925 assessed in hNR1-expressing HEK cells transfected with habituation sessions with at least 30 minutes between each ses- hNR2A, 2B, 2C, and 2D are shown in Figure 2. The activity of NYX- sion and an additional habituation session immediately before 2925 was 40.6%, 47.1%, 63.1%, and 57.8% of [ H] MK-801 activity dosing (0 min). Animals were dosed with NYX-2925 (1–100 mg/ measured in the presence of maximal glycine for hNR2A, 2B, 2C, kg p.o.) or CMC saline. Animals were tested 15, 30, 60, and 120 and 2D receptors, respectively. The potency (EC) of NYX-2925 Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 247 Figure 2.NYX-2925 is active at all 4 human N-methyl-D-aspartate receptor (hNMDAR) 2 subtypes. Potentiation of [ H] MK-801 binding by NYX-2925 and glycine in hNM- DAR subtype-expressing human embryonic kidney (HEK) cells. Stable hNMDAR1-expressing HEK cells were transiently transfected with cDNAs encoding hNMDAR2A, hNMDAR2B, hNMDAR2C, or hNMDAR2D. At 48 hours post-transfection, membrane-bound receptors were isolated, and the functional glycine site agonist effects were measured using the [ H] MK-801 potentiation assay, as described in Methods. The percent maximal effects were calculated relative to stimulation in the presence of 1 mM glycine and 50 μM glutamate. Binding curves were fitted using GraphPad Prism software. was 55 pM, 28 fM, 11 pM, and 55 pM for hNR2A, 2B, 2C, and 2D [5000 nM] vs vehicle, P < .05; Figure  3C). Moreover, LTP at mixed receptors, respectively. excitatory inputs synapsing in layer II/III of MPFC was also facili- NYX-2925 (10 µM) did not exhibit any significant affinity (con- tated by NYX-2925 (F = 5.6, P < .05; Fisher’s PLSD posthoc test (4,27) sidered >50%) for any of the 81 binding sites tested (Table). 1 At 10 NYX-2925 [100 and 500 nM] vs vehicle, P < .05; Figure 3D). μM, NYX-2925 did not demonstrate significant inhibition or stim- ulation (agonism) in any of the 81 sites. This study provides evi- Pharmacokinetics and Toxicology dence that NYX-2925 has a low potential for “off-target” activity. NYX-2925 showed high oral bioavailability, CNS penetration, and had a wide therapeutic index. As shown in Figure 4A, NYX-2925 NMDAR Current and Long-Term Potentiation in (1 mg/kg p.o.) has a plasma Cmax of 706 nM at 1 hour, a plasma Hippocampus and Medial Prefrontal Cortex half-life of 6.8 hours, a CSF Cmax of 44 nM at 1 hour, and a CSF NYX-2925 (100–500 nM) increased the magnitude of pharmaco- half-life of 1.2 hours. The Cmax CSF exposure is 44 nM. The oral logically isolated NMDA current in Schaffer collateral-evoked bioavailability of NYX-2925 in plasma is 56% as calculated by the EPSCs in CA1 pyramidal neurons (repeated-measures ANOVA area under the curve of plasma exposure following 2 mg/kg i.v. F = 7.2, P < .05; within-subjects t test vs baseline NYX-2925 or 10 mg/kg p.o. dosing (mean ± SEM AUC IV 2155.12 ± 45.77, p.o. (3,18) [100 and 500 nM] vs vehicle, P < .05; Figure  3A). NYX-2925 also 6053.73 ± 498.55 ng.h/mL). increased the magnitude of LTP at Schaffer collateral-CA1 syn- The no observed adverse effect level of NYX-2925 for male apses (F = 6.2, P < .05; Fisher’s PLSD posthoc test NYX-2925 [500 Sprague Dawley rats when administered by the oral (gavage) (4,30) nM] vs vehicle, P <.05; F igure 3B) and inhibited LTD at these same route, once daily for 14 consecutive days is 1000  mg/kg/d as synapses (F = 4.1, P < .05; Fisher’s PLSD posthoc test NYX-2925 measured by mortality, clinical signs, body weight, food intake, (3,28) Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 248 | International Journal of Neuropsychopharmacology, 2018 Table 1. Radioligand Displacement by NYX-2925 (10 μM) Table 1. Continued NYX-2925 NYX-2925 Site % Inhibition Site % Inhibition A1 (antagonist) -4.2 5-HT2C (antagonist) -5.9 A2A (agonist) -6.1 5-HT3 (antagonist) 2.7 A3 (agonist) -4.9 5-HT5a (agonist) -7.5 α1 non-selective (antagonist) -10 5-HT6 (agonist) -8.6 α2 non-selective (antagonist) -11.6 5-HT7 (agonist) -5.1 β1 (agonist) 4.6 Sigma (non-selective) (agonist) -15.8 β2 (agonist) -7.6 SSST (non-selective) (agonist) -13.8 AT1 (antagonist) -0.8 GR (agonist) -6.2 AT2 (agonist) 1.8 VPAC1 (VIP1) (agonist) 7.4 BZD central (agonist) -9.1 V1 a (agonist) 15.3 BZD peripheral (antagonist) -6.6 Verapamil site (antagonist) 3.4 BB non-selective (agonist) -9 hERG [3H] Dofetilide -3 B2 (agonist) -3.1 KV channel (antagonist) -7.4 CGRP (agonist) 3.6 SKCa channel (antagonist) 5 CB1 (agonist) 5.7 Na+ channel (site 2) (antagonist) 7.3 CCK1 CCKA (agonist) 3.8 Cl- channel GABA-gated (antagonist) 5.2 CCK2 CCKB (agonist) -13 Norepinephrine transporter (antagonist) -6.2 D1 (antagonist) -19.9 Dopamine transporter (antagonist) -6.4 D2S (antagonist) 6.8 5-HT transporter (antagonist) -13.9 D3 (antagonist) -5.2 Results that show an inhibition or stimulation >50% D4.4 (antagonist) -5 are considered to represent significant effects of the test compound. D5 (antagonist) 4.1 ETA (agonist) 5.7 ETB (agonist) 1.3 and gross histopathology as well as organ weights. The plasma GABA non-selective (agonist) -18.8 exposure (area under the curve) of NYX-2925 following 14 daily GAL1 (agonist) 3 doses of 1000  mg/kg was 1 074 478  ng.h/mL compared with GAL2 (agonist) 3.7 494 ng.h/mL for a single behaviorally efficacious dose of 1 mg/ PDGF (agonist) -2.6 kg, thus resulting in a projected therapeutic index of 2175. CXCR2 IL-8B (agonist) -4.5 CCR1 (agonist) 0 Structural Plasticity TNF-α (agonist) -21.7 H1 (antagonist) 2.4 NYX-2925 (1  mg/kg p.o.; 24 hours post dosing) induced struc- H2 (antagonist) 0.5 tural plasticity as indexed by increased diameter of spine heads MC4 (agonist) 3.4 (F = 44.2, P < .05) and necks (F = 27.2, P < .05) in the pri- (1,3441) (1,3441) MT1 ML1A (agonist) 1.4 mary apical dendrites of dentate granule neurons (Figure  4B). M1 (antagonist) -23.4 Spine length (F = 2.9, P > .05) or total spine number (F = 1.1, (1,3441) (1,65) M2 (antagonist) -6.1 P > .05) was not affected by NYX-2925 (Figure 4B). M3 (antagonist) -10.1 M4 (antagonist) -11.3 M5 (antagonist) 5.6 Metaplasticity NK1 (agonist) -22.5 NYX-2925 persistently enhanced LTP at 24 hours and 1 week NK2 (agonist) 2.4 following a single dose (1 mg/kg p.o.) at Schaffer collateral-CA1 NK3 (antagonist) -7.7 synapses after 3 submaximal high-frequency stimulus trains Y1 (agonist) -3.4 Y2 (agonist) -9.9 (2 × 100 Hz/800  ms, arrows). LTP was evaluated either 24 hours NTS1 / NT1 (agonist) -11.4 post dosing (F = 5.5, P < .05) or 1 week post dosing (F = 8.8, (1,13) (1,14) δ2 (DOP) (agonist) -4.2 P < .05) as shown in Figure 4C-D. κ (KOP) (agonist) 0.1 μ (MOP) (agonist) -4.5 Learning and Memory Tests NOP / ORL1 (agonist) 5.9 PAC1 / PACAP (agonist) -4.4 NYX-2925 facilitated learning and memory in multiple para- PPARγ (agonist) -10.7 digms across a wide dose range. NYX-2925 (0.01, 0.1, 1  mg/kg PCP (antagonist) -5.7 p.o.), delivered 1 hour prior to T1, increased novel object rec- EP2 (agonist) 4.7 ognition after a 24-hour delay between training and testing to EP4 (agonist) 7.5 a similar degree as the positive control SB399885, delivered 4 IP PGI2 (agonist) -5.1 hours prior to T1 and T2 (F = 11.5, P < .05; Fisher’s PLSD post- (4,54) P2X (agonist) 16.1 hoc test NYX-2925 [0.01, 0.1, 1  mg/kg] or SB399885 vs vehicle, P2Y (agonist) 5.1 P < .05; Figure 5A). 5-HT1A (agonist) 2.5 NYX-2925 (1  mg/kg p.o.) facilitated positive emotional 5-HT1B (antagonist) -6.7 learning and this effect was blocked by pretreatment with 5-HT2A (antagonist) -5.8 a single dose of the NMDAR antagonist CPP as measured by 5-HT2B (agonist) 2.1 both increased rates of hedonic 50-kHz USVs across trials Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 249 Figure 3. NYX-2925 facilitates NMDA current and NMDA receptor-dependent LTP, while decreasing LTD. (A) NYX-2925 (0.1–0.5 µM) enhanced the pharmacologically- isolated NMDAR current in Schaffer collateral-evoked EPSCs in hippocampal CA1 pyramidal neurons. NYX-2925 (B) enhanced the magnitude of LTP at 0.5 µM and (C) decreased the magnitude of LTD at 5 µM of synaptic transmission at Schaffer collateral-CA1 synapses as compared to untreated control (aCSF) slices. (D) NYX-2925 (0.1 and 0.5 µM) enhanced the magnitude of LTP in layer II/III-evoked normalized field EPSP slopes in rat slices recorded in MPFC layer IV. NYX-2925 was bath applied for 40–50 min starting 20 min before high-frequency theta trains to induce LTP (solid bar) or low-frequency stimulation to elicit LTD (solid bar). LTP/LTD was measured 40 min after high- or low-frequency stimulation. Data are shown as mean ± SEM. N = 7 cells per gr oup (A) or 5–10 slices per group (B-D). * P.05, < (A) within-subjects t test vs baseline (B-D) Fishers PLSD post hoc test vs vehicle. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 250 | International Journal of Neuropsychopharmacology, 2018 Figure 4. NYX-2925 persistently enhances hippocampal structural plasticity and metaplasticity following a single in vivo dose (1 mg/kg PO). (A) Plasma and CSF levels following dosing with NYX-2925 (1 mg/kg PO) were measured by LC/MS/MS. The C in the CSF is 44 nM (at 30 min post-dosing) which is well above the threshold for max activing NR2A-D receptors (1 nM) as shown in Figure 2. A single in vivo dose of NYX-2925 (1 mg/kg PO) increased (B) spine head and neck diameter in hippocampal dentate gyrus primary dendrites 24 hrs post-dosing, and (C,D) the magnitude of LTP compared to slices from vehicle-treated control rats prepared (C) 24 hrs and (D) 1 week post-dosing (NYX-2925, 1 mg/kg PO) at Schaffer collateral-CA1 synapses after 3 sub-maximal high-frequency stimulus trains (2 × 100 Hz/800 ms, arrows). Data are shown as Mean ± SEM. N = (A) 2–3 rats, (B) 1412–2034 spines, and (C-D) 6–8 slices per group. (repeated-measures ANOVA: dosing group F = 37.7, P < .05; drug discrimination task (F = 90.9, P < .05; Fisher’s PLSD posthoc (3,31) (6,36) trials F = 39.8, P < .05, dosing group x trials F = 15.0, test ketamine [17 and 30 mg/kg p.o.] vs vehicle, P < .05; Figure  6C). (5,31) (15,31) P < .05, Fisher’s PLSD posthoc test NYX-2925 alone vs all other Unlike ketamine, NYX-2925 did not suppress operant responding groups, P < .05; Figure  5B) and increased running speed to self- at the highest dose tested (F = 9.1, P < .05; Fisher’s PLSD post hoc (6,36) administer heterospecific rough-and-tumble play (F = 12.6, test ketamine [30 mg/kg p.o.] vs vehicle, P < .05; F igure 6D)). (3,31) P < .05; Fisher’s PLSD posthoc test NYX-2925 alone vs all other groups, P < .05; Figure  5C). In addition, NYX-2925 (0.001–1  mg/ Discussion kg p.o.) increased positive emotional learning 1 hour post dos- The NMDA receptor has recently become an attractive target for ing (F = 4.6, P < .05, Fisher’s PLSD posthoc test 0.001, 0.01, 0.1, (5,53) the development of therapeutics for the treatment of a variety of 1 mg/kg vs vehicle, P < .05; data not shown). CNS disorders. Clinical trial data have shown that NMDA receptor modulators have efficacy in mood disorders such as depression, Safety Pharmacology obsessive compulsive disorder, schizophrenia, as well as in neuro- NYX-2925 did no induce sedation/ataxia and does not show pathic pain, among others (Rodriguez et al., 2013 Cain et  ; al., 2014; ketamine-like drug discrimination. NYX-2925 (1–100 mg/kg Feder et al., 2014; Fond et al., 2014; Maher et al., 2017). To date, only p.o.) did not induce a sedative/ataxic effect in the Rota-Rod test rapastinel and DCS have shown efficacy in clinical trials without (F = 0.5, P > .05; Figure 6A) or alter locomotor activity in the the side effects seen with NMDA receptor antagonists. DCS has (3,29) open field test at 1 mg/kg p.o. (F = 0.002, P > .05; Figure 6B). shown a comparatively weak therapeutic response profile and (1,22) Ketamine (p.o.), but not NYX-2925, substituted for a training results have been mixed (Laake and Oeksengaard, 2002; Tuominen dose of ketamine (5.6 mg/kg i.p.) in a dose-dependent manner in the et al., 2005; Ori et al., 2015). Rapastinel is a peptide that must be Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 251 Figure 5. NYX-2925 facilitates learning and memory by activating NMDA receptors. (A) NYX-2925 (0.1–1 mg/kg PO, delivered 1 h prior to training) facilitated novel object recognition as measured by the D2 index [(novel object exploration time – familiar object exploration time) / total exploration time in T2)] tested 24 hrs after training. Pretreatment with a silent dose of the NMDA receptor antagonist CPP blocked the facilitation of learning in the positive emotional learning assay seen with NYX-2925 tested 1 hr after NYX-2925 administration (1 mg/kg PO) as measured by (B) increased rates of hedonic 50-kHz USVs across trial blocks and (C) increased running speed to self-administer heterospecific rough-and-tumble play. Data are shown as mean ± SEM. N = rats 8–12 per group. * P < .05, Fishers PLSD post hoc test (A) vs vehicle or (B) vs all other groups. Figure 6. NYX-2925 does not produce ketamine-like sedative/ataxic or discriminative stimulus effects. (A) NYX-2925 (1, 10, 100 mg/kg PO) did not produce sedation/ ataxia in the fixed speed (16 RPM) RotaRod test. (B) Locomotor activity in the open field was not altered by NYX-2925 (1 mg/kg PO). (C,D) NYX-2925 does not substitute for ketamine as measured by (C) percentage ketamine-lever responding and (D) rates of responding for ketamine (10, 17, 30 mg/kg PO) and NYX-2925 (1, 10, 100 mg/kg PO) in rats trained to discriminate 5.6 mg/kg ketamine from saline. N = 6–12 per group. * Fisher’s PLSD post hoc test vs vehicle. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 252 | International Journal of Neuropsychopharmacology, 2018 administered i.v. To obviate these problems, we developed a pep- D. R. Houck, A. L. Gross, C. Cearley, T. M. Madsen, R. A. Kroes, J. S. tide mimetic program that uses spirocyclic--lactam β chemistry to Burgdorf, and J.  R. Moskal are employees of Aptinyx, Inc., and mimic the dipyrrolidine core of rapastinel. have received financial compensation and stock. The data reported here show that NYX-2925 is an NMDAR modulator that enhances activity-dependent synaptic plasticity in both the hippocampus and MPFC and facilitates learning and Acknowledgments memory. This suggests that NYX-2925, like rapastinel (Moskal The authors thank Puja Kansara, Elizabeth Pollard, Emma et al., 2017), operates through an NMDAR-triggered process that Rodriguez, and Mary Schmidt for their excellent technical leads to lowering of the threshold for induction of LTP that results assistance. in enhancement of learning and memory, as well as long-term metaplasticity that persistently shifts the threshold for induction of LTP. In vivo, NYX-2925 facilitated novel object recognition and References positive emotional learning, both of which are NMDAR-dependent learning tasks (Burgdorf et al., 2011b v; an der Staay et al., 2011), Abraham WC, Bear MF (1996) Metaplasticity: the plasticity of and these effects were blocked by pretreatment with the NMDAR synaptic plasticity. Trends Neurosci 19:126–130. glutamate site antagonist CPP. Further, the dose used to facilitate Alonso E, Lopez-Ortiz F, del Pozo C, Peralta E, Macias A, Gonzalez learning in vivo (1 mg/kg p.o.) led to CSF drug levels that activate J (2001) Spiro beta-lactams as beta-turn mimetics. Design, NR2A-D containing NMDARs in vitro, facilitate both NMDA cur - synthesis, and NMR conformational analysis. J Org Chem rent and NMDAR-dependent LTP in slices from hippocampus and 66:6333–6338. MPFC, and reduce while actually reducing LTD in hippocampus. Bartlett TE, Bannister NJ, Collett VJ, Dargan SL, Massey PV, The observation that NYX-2925 shares with rapastinel an abil- Bortolotto ZA, Fitzjohn SM, Bashir ZI, Collingridge GL, Lodge D ity to simultaneously enhance the induction of LTP, while sup- (2007) Differential roles of NR2A and NR2B-containing NMDA pressing that of LTD (X. L. Zhang et al., 2008), is intriguing, given receptors in LTP and LTD in the CA1 region of two-week old that studies have suggested that LTP and LTD may be preferen- rat hippocampus. Neuropharmacology 52:60–70. tially induced by NMDARs containing NR2B and NR2A subunits, Bittermann H, Gmeiner P (2006) Chirospecific synthesis of spiro- respectively (Bartlett et al., 2007 Morishita et al., ; 2007Y ; ashiro cyclic beta-lactams and their characterization as potent type and Philpot, 2008). This leads us to conclude that NYX-2925, like II beta-turn inducing peptide mimetics. J Org Chem 71:97–102. rapastinel, may selectively activate NR2B-containing NMDARs Bliss TV, Collingridge GL (1993) A synaptic model of mem- (Zhang et al., 2008). In addition, NYX-2925 facilitated learning and ory: long-term potentiation in the hippocampus. Nature memory, metaplasticity of LTP, and structural plasticity, 24 hours 361:31–39. after a single dose, when NYX-2925 was no longer present in the Burgdorf J, Zhang XL, Weiss C, Matthews E, Disterhoft JF, Stanton CSF. Thus, NYX-2925 produces its behavioral effects by facilitat- PK, Moskal JR (2011a) The N-methyl-D-aspartate recep- ing NMDAR-dependent plasticity acutely via direct activation of tor modulator GLYX-13 enhances learning and memory, in NMDAR and chronically by enhancing both metaplasticity and young adult and learning impaired aging rats. Neurobiol structural plasticity triggered by NMDAR activation. Aging 32:698–706. NYX-2925 displays the properties of an attractive therapeutic Burgdorf J, Kroes RA, Weiss C, Oh MM, Disterhoft JF, Brudzynski for NMDAR-modulated CNS disorders. It operates through an SM, Panksepp J, Moskal JR (2011b) Positive emotional learn- NMDAR-triggered, AMPAR-dependent mechanism that leads to ing is regulated in the medial prefrontal cortex by GluN2B- metaplasticity processes similar to LTP, which result in enhance- containing NMDA receptors. Neuroscience 192:515–523. ments in learning and memory as well as long-term metaplasticity Burgdorf J, Zhang XI, Nicholson KL, Balster RL, Leander JD, (Abraham and Bear, 1996). NYX-2925 is orally bioavailable and does Stanton PK, Gross AL, Kroes RA, Moskal JR (2013) GLYX-13, not show ketamine-like side effects or observable adverse effects an NMDA receptor glycine-site functional partial agonist, in toxicology studies, suggesting a wide therapeutic index (>1000). induces antidepressant-like effects without ketamine-like Alonso et  al. (2001) showed that spirocyclic -lactams could β side effects. Neuropsychopharmacology 38:729–742. be synthesized with β -turn conformations making the creation Burgdorf J, Kroes RA, Zhang XL, Gross AL, Schmidt M, Weiss of peptidomimetics using conventional peptide chemistry tech- C, Disterhoft JF, Burch RM, Stanton PK, Moskal JR (2015a) niques possible, and Bittermann and Gmeiner (2006) reported the Rapastinel (GLYX-13) has therapeutic potential for the synthetic methods for β-turn-containing spirocyclic- -lactams β treatment of post-traumatic stress disorder: characteriza- starting from natural proline. Recently, this approach has been tion of a NMDA receptor-mediated metaplasticity process used to develop peptidomimetics of the dopamine receptor mod- in the medial prefrontal cortex of rats. Behav Brain Res ulating peptide L-prolyl-L-leucyl-glycinamide (Khalil et al., 1999) 294:177–185. and the creation of somatostatin mimetics (Lesma et  al., 2013). Burgdorf J, Zhang XL, Weiss C, Gross A, Boikess SR, Kroes RA, Using a similar chemical synthesis approach, we have been able Khan MA, Burch RM, Rex CS, Disterhoft JF, Stanton PK, Moskal to create a novel platform of rapastinel mimetics, exemplified by JR (2015b) The long-lasting antidepressant effects of rapasti- NYX-2925 that may be useful tools to study NMDAR structure and nel (Glyx-13) are associated with a metaplasticity process in function. And since NYX 2925 appears to be an excellent thera- the medial prefrontal cortex and hippocampus. Neuroscience peutic candidate, other compounds from this platform may lead 308:202–211. to additional NMDAR modulators with therapeutic potential. Burgdorf J, Colechio EM, Stanton P, Panksepp J (2017) Positive emotional learning induces resilience to depression: a role for NMDA receptor-mediated synaptic plasticity. Curr Statement of Interest Neuropharmacol 15:3–10. P. K. Stanton is a consultant for Aptinyx, Inc. and has received Cain CK, McCue M, Bello I, Creedon T, Tang DI, Laska E, Goff DC financial compensation and stock. X.-L. Zhang is supported by (2014) d-Cycloserine augmentation of cognitive remediation a grant from Aptinyx, Inc., granted to P. K. Stanton. M. A. Khan, in schizophrenia. Schizophr Res 153:177–183. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Khan et al. | 253 Coyle JT (2012) NMDA receptor and schizophrenia: a brief his- Lesma G, Cecchi R, Cagnotto A, Gobbi M, Meneghetti F, Musolino tory. Schizophr Bull 38:920–926. M, Sacchetti A, Silvani A (2013) Tetrahydro-beta-carboline- Cull-Candy S, Brickley S, Farrant M (2001) NMDA receptor subu- based spirocyclic lactam as type II’ beta-turn: application nits: diversity, development and disease. Curr Opin Neurobiol to the synthesis and biological evaluation of somatostatine 11:327–335. mimetics. J Org Chem 78:2600–2610. Danysz W, Parsons CG (1998) Glycine and N-methyl-D-aspartate Lu W, Du J, Goehring A, Gouaux E (2017) Cryo-EM structures of receptors: physiological significance and possible therapeutic the triheteromeric NMDA receptor and its allosteric modula- applications. Pharmacol Rev 50:597–664. tion. Science 355:pii: eaal3729. de Kleine RA, Hendriks GJ, Kusters WJ, Broekman TG, van Maher DP, Chen L, Mao J (2017) Intravenous ketamine infusions Minnen A (2012) A randomized placebo-controlled trial of for neuropathic pain management: a promising therapy in D-cycloserine to enhance exposure therapy for posttrau- need of optimization. Anesth Analg 124:661–674. matic stress disorder. Biol Psychiatry 71:962–968. Matsuzaki M, Honkura N, Ellis-Davies GC, Kasai H (2004) Dolino DM, Cooper D, Ramaswamy S, Jaurich H, Landes CF, Structural basis of long-term potentiation in single dendritic Jayaraman V (2015) Structural dynamics of the glycine- spines. Nature 429:761–766. binding domain of the N-methyl-D-aspartate receptor. J Biol Millecamps M, Centeno MV, Berra HH, Rudick CN, Lavarello S, Chem 290:797–804. Tkatch T, Apkarian AV (2007) D-cycloserine reduces neuro- Feder A, Parides MK, Murrough JW, Perez AM, Morgan JE, Saxena pathic pain behavior through limbic NMDA-mediated cir - S, Kirkwood K, Aan Het Rot M, Lapidus KA, Wan LB, Iosifescu cuitry. Pain 132:108–123. D, Charney DS (2014) Efficacy of intravenous ketamine for Morishita W, Lu W, Smith GB, Nicoll RA, Bear MF, Malenka RC treatment of chronic posttraumatic stress disorder: a rand- (2007) Activation of NR2B-containing NMDA receptors is not omized clinical trial. JAMA Psychiatry 71:681–688. required for NMDA receptor-dependent long-term depres- Fond G, Loundou A, Rabu C, Macgregor A, Lancon C, Brittner M, sion. Neuropharmacology 52:71–76. Micoulaud-Franchi JA, Richieri R, Courtet P, Abbar M, Roger Morris RG (2013) NMDA receptors and memory encoding. M, Leboyer M, Boyer L (2014) Ketamine administration in Neuropharmacology 74:32–40. depressive disorders: a systematic review and meta-analysis. Moskal JR, Burgdorf JS, Stanton PK, Kroes RA, Disterhoft JF, Burch Psychopharmacology (Berl) 231:3663–3676. RM, Khan MA (2017) The development of rapastinel (formerly Ghasemi M, Phillips C, Trillo L, De Miguel Z, Das D, Salehi A (2014) GLYX-13); a rapid acting and long lasting antidepressant. Curr The role of NMDA receptors in the pathophysiology and treat- Neuropharmacol 15:47–56. ment of mood disorders. Neurosci Biobehav Rev 47:336–358. Moskal JR, Kuo AG, Weiss C, Wood PL, O’Connor Hanson A, Kelso Ghasemi M, Schachter SC (2011) The NMDA receptor complex S, Harris RB, Disterhoft JF (2005) GLYX-13: a monoclonal anti- as a therapeutic target in epilepsy: a review. Epilepsy Behav body-derived peptide that acts as an N-methyl-D-aspartate 22:617–640. receptor modulator. Neuropharmacology 49:1077–1087. Goff DC (2012) D-cycloserine: an evolving role in learning and Nadeson R, Tucker A, Bajunaki E, Goodchild CS (2002) Potentiation neuroplasticity in schizophrenia. Schizophr Bull 38:936–941. by ketamine of fentanyl antinociception. I. An experimental Haring R, Stanton PK, Scheideler MA, Moskal JR (1991) Glycine- study in rats showing that ketamine administered by non- like modulation of N-methyl-D-aspartate receptors by a spinal routes targets spinal cord antinociceptive systems. Br monoclonal antibody that enhances long-term potentiation. J Anaesth 88:685–691. J Neurochem 57:323–332. Noguchi J, Matsuzaki M, Ellis-Davies GC, Kasai H (2005) Spine- Harris RE, Sundgren PC, Pang Y, Hsu M, Petrou M, Kim SH, neck geometry determines NMDA receptor-dependent Ca2+ McLean SA, Gracely RH, Clauw DJ (2008) Dynamic levels of signaling in dendrites. Neuron 46:609–622. glutamate within the insula are associated with improve- Ori R, Amos T, Bergman H, Soares-Weiser K, Ipser JC, Stein DJ ments in multiple pain domains in fibromyalgia. Arthritis (2015) Augmentation of cognitive and behavioural therapies Rheum 58:903–907. (CBT) with d-cycloserine for anxiety and related disorders. Hirst WD, Stean TO, Rogers DC, Sunter D, Pugh P, Moss SF, Cochrane Database Syst Rev:CD007803. Bromidge SM, Riley G, Smith DR, Bartlett S, Heidbreder CA, Ota KT, Liu RJ, Voleti B, Maldonado-Aviles JG, Duric V, Iwata M, Atkins AR, Lacroix LP, Dawson LA, Foley AG, Regan CM, Upton Dutheil S, Duman C, Boikess S, Lewis DA, Stockmeier CA, N (2006) SB-399885 is a potent, selective 5-HT6 receptor antag- DiLeone RJ, Rex C, Aghajanian GK, Duman RS (2014) REDD1 onist with cognitive enhancing properties in aged rat water is essential for stress-induced synaptic loss and depressive maze and novel object recognition models. Eur J Pharmacol behavior. Nat Med 20:531–535. 553:109–119. Paoletti P, Bellone C, Zhou Q (2013) NMDA receptor subunit div -er Iacobucci GJ, Popescu GK (2017) NMDA receptors: linking physio- sity: impact on receptor properties, synaptic plasticity and logical output to biophysical operation. Nat Rev Neurosci disease. Nat Rev Neurosci 14:383–400. 18:236–249. Parent MA, Wang L, Su J, Netoff T, Yuan LL (2010) Identification of Ishiyama S, Brecht M (2016) Neural correlates of ticklishness in the hippocampal input to medial prefrontal cortex in vitro. the rat somatosensory cortex. Science 354:757–760. Cereb Cortex 20:393–403. Karakas E, Furukawa H (2014) Crystal structure of a heterotetra- Patrizi A, Picard N, Simon AJ, Gunner G, Centofante E, Andrews NA, meric NMDA receptor ion channel. Science 344:992–997. Fagiolini M (2016) Chronic administration of the N-methyl- Khalil EM, Ojala WH, Pradhan A, Nair VD, Gleason WB, Mishra D-aspartate receptor antagonist ketamine improves Rett RK, Johnson RL (1999) Design, synthesis, and dopamine Syndrome phenotype. Biol Psychiatry 79:755–764. receptor modulating activity of spiro bicyclic peptid- Preskorn S, Macaluso M, Mehra DO, Zammit G, Moskal JR, Burch omimetics of L-prolyl-L-leucyl-glycinamide. J Med Chem RM, Group GCS (2015) Randomized proof of concept trial of 42:628–637. GLYX-13, an N-methyl-D-aspartate receptor glycine site par - Laake K, Oeksengaard AR (2002) D-cycloserine for Alzheimer’s tial agonist, in major depressive disorder nonresponsive to a disease. Cochrane Database Syst Rev:CD003153. previous antidepressant agent. J Psychiatr Pract 21:140–149. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018 254 | International Journal of Neuropsychopharmacology, 2018 Pyke T, Osmotherly PG, Baines S (2016) Measuring glutamate lev- van der Staay FJ, Rutten K, Erb C, Blokland A (2011) Effects of the els in the brains of fibromyalgia patients and a potential role cognition impairer MK-801 on learning and memory in mice for glutamate in the pathophysiology of fibromyalgia symp- and rats. Behav Brain Res 220:215–229. toms: a systematic review. Clin J Pain 33:944–954. Vasilescu AN, Schweinfurth N, Borgwardt S, Gass P, Lang Robb CM (1991) Restrictive covenant law in Georgia: back to the UE, Inta D, Eckart S (2017) Modulation of the activity of drawing board. J Med Assoc Ga 80:546–548. N-methyl-d-aspartate receptors as a novel treatment Rodriguez CI, Kegeles LS, Levinson A, Feng T, Marcus SM, option for depression: current clinical evidence and thera- Vermes D, Flood P, Simpson HB (2013) Randomized con- peutic potential of rapastinel (GLYX-13). Neuropsychiatr Dis trolled crossover trial of ketamine in obsessive-compulsive Treat 13:973–980. disorder: proof-of-concept. Neuropsychopharmacology 38: Wilkinson D, Wirth Y, Goebel C (2014) Memantine in patients 2475–2483. with moderate to severe Alzheimer’s disease: meta-analyses Rodriguez CI, Zwerling J, Kalanthroff E, Shen H, Filippou M, Jo using realistic definitions of response. Dement Geriatr Cogn B, Simpson HB, Burch RM, Moskal JR (2016) Effect of a novel Disord 37:71–85. NMDA receptor modulator, rapastinel (formerly GLYX-13), in Yashiro K, Philpot BD (2008) Regulation of NMDA receptor sub- OCD: proof of concept. Am J Psychiatry 173:1239–1241. unit expression and its implications for LTD, LTP, and meta- Tavoloni N, Schaffner F (1989) Bile secretory apparatus in the plasticity. Neuropharmacology 55:1081–1094. newborn dog: relationship between structural and functional Zhang L, Xu T, Wang S, Yu L, Liu D, Zhan R, Yu SY (2013) NMDA immaturities. Biol Neonate 55:124–135. GluN2B receptors involved in the antidepressant effects of cur - Thompson LT, Moskal JR, Disterhoft JF (1992) Hippocampus- cumin in the forced swim test. Prog Neuropsychopharmacol dependent learning facilitated by a monoclonal antibody or Biol Psychiatry 40:12–17. D-cycloserine. Nature 359:638–641. Zhang XL, Sullivan JA, Moskal JR, Stanton PK (2008) A NMDA Traynelis SF, Wollmuth LP, McBain CJ, Menniti FS, Vance KM, receptor glycine site partial agonist, GLYX-13, simultan- Ogden KK, Hansen KB, Yuan H, Myers SJ, Dingledine R (2010) eously enhances LTP and reduces LTD at Schaffer collat- Glutamate receptor ion channels: structure, regulation, and eral-CA1 synapses in hippocampus. Neuropharmacology function. Pharmacol Rev 62:405–496. 55:1238–1250. Tuominen HJ, Tiihonen J, Wahlbeck K (2005) Glutamatergic drugs Zhou HY, Chen SR, Pan HL (2011) Targeting N-methyl-D-aspartate for schizophrenia: a systematic review and meta-analysis. receptors for treatment of neuropathic pain. Expert Rev Clin Schizophr Res 72:225–234. Pharmacol 4:379–388. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/3/242/4584012 by Ed 'DeepDyve' Gillespie user on 16 March 2018

Journal

International Journal of NeuropsychopharmacologyOxford University Press

Published: Mar 1, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off