Animals.

Studies were conducted at either Bristol-Myers Squibb (BMS, Wallingford, CT) or at the Biocon Bristol-Myers Squibb Research Center (BBRC, Bangalore, India).

BMS Studies.

Animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited facility and maintained in accordance with the guidelines of the Animal Care and Use Committee of the Bristol-Myers Squibb Company, the “Guide for Care and Use of Laboratory Animals,” and the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Research protocols were approved by the Bristol-Myers Squibb Company Animal Care and Use Committee. In vivo [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([3H]MK-801) binding studies were conducted in singly housed, male Sprague Dawley rats (170–250 g; ∼6–8 weeks of age) supplied with a previously implanted jugular vein catheter (Hilltop Laboratory Animals Inc., Scottsdale, PA). Ex vivo GluN2B occupancy studies were conducted in male Sprague Dawley rats housed two rats/cage purchased from Charles River Laboratories (Raleigh, NC; 5–7 weeks of age) or Harlan (Indianapolis, IN; 7–9 weeks of age). Ex vivo long-term potentiation (LTP) studies were conducted in male Sprague Dawley rats housed two rats/cage purchased from Harlan (4–6 weeks of age). Cage size for both single- and pair-housed rats was 10.25 × 14 inches, and animals were housed in temperature- (21 ± 2°C) and humidity-controlled (50% ± 10%) rooms maintained under a 12-hour light/dark cycle (lights on at 06:00 hours) with food and water available ad libitum. Cognitive performance, behavioral observational studies, and quantitative electroencephalogram (qEEG) studies were conducted in separate cohorts of male cynomolgus monkeys (Macaca fascicularis; 5–7 years of age, 5.0–8.5 kg). Monkeys were typically pair housed [cage size (width/depth/height): 64 × 31 × 33 inches] and fed standard monkey chow (Teklad global 20% protein Primate Diet 2050; Harlan). Subjects for cognitive testing were fed sufficient quantities to ensure normal growth while maintaining motivation to perform cognitive tasks (Weed et al., 1999); subjects for behavioral observation were food restricted overnight prior to compound administration. For all subjects, water was continuously available except during studies, and fresh fruit or dietary enrichment was provided twice weekly. Toys and foraging devices were routinely provided, and television programs were available in the colony rooms. Subjects were fitted with plastic or metal neck collars (Primate Products, Immokalee, FL).

BBRC Studies.

All experimental procedures were conducted in accordance with the guidelines set by the Committee for the Purpose of Control and Supervision on Experiments on Animals, Ministry of Environment and Forests, Government of India. An institutional animal ethics committee approved the experimental protocols, and all animals were housed in an Association for Assessment and Accreditation of Laboratory Animal Care–accredited animal facility. Female Xenopus laevis were obtained from Nasco (Fort Atkinson, WI) and housed two frogs/cage in Tecniplast (Buguggiate, Varese, Italy) XenopLus standalone housing. Water temperature and pH were maintained at 18 ± 1°C and 6.8–7.4, respectively, with regular monitoring of nitrogenous wastes. Frogs were fed with Zeigler adult Xenopus diet (Xenopus Express, Brooksville, FL) three times per week, and each cage was provided with enrichment tubes and pads. Male Sprague Dawley rats (7–10 weeks of age; Harlan, Livermore, CA) were used for in vitro GluN2B binding studies and were housed three rats/cage (cage size: 12.6 × 12 inches). Male CD-1 mice were obtained directly from Harlan (Horst, Limburg, Netherlands) for GluN2B occupancy (5–8 weeks of age) and locomotor activity (LMA) studies (6–7 weeks of age) or from in-house breeding colonies derived from Harlan CD-1 mice for the forced swim test (FST; 6–7 weeks of age). Novelty suppressed feeding (NSF) studies were conducted in male BALB/c mice (13–14 weeks of age; Taconic Vivo-bio, Hyderabad, India). Mice were housed five/cage (cage size: 12.8 × 6.6 inches), and all rodents were held in colony rooms with controlled temperature (22–24°C) and humidity (48%–54%) under a 12-hour light/dark cycle (lights on at 07:00 hours) with food and water available ad libitum unless specified otherwise.

Reagents.

BMS-986169, BMS-986163, CP-101,606 [Traxoprodil; free base (in vitro studies) or methane sulfonic acid salt], Ro 25-6981, MK-0657 (CERC-301; 4-methylbenzyl-(3S,4R)-3-fluoro-4-[(2-pyrimidinylamino)methyl]-1-piperidinecarboxylate), [³H]Ro 25-6981, and [³H]MK-801 were synthesized by the BMS or BBRC chemistry groups or the BMS radiosynthesis group. The chemical structures of BMS-986169 and the phosphate prodrug BMS-986163 are shown in Fig. 1. Sources of other reagents were as follows: (+)MK-801 hydrogen maleate (Sigma-Aldrich, St. Louis, MO), ketamine HCl [Ketaset (Fort Dodge Animal Health, Fort Dodge, IA) or Aneket (Neon Pharmaceuticals, Mumbai, India)], 1-[2-(4-chlorophenyl)ethyl]-1,2,3,4-tetrahydro-6-methoxy-2-methyl-7-isoquinolinol hydrochloride (Ro-04-5595) (Tocris Bioscience, Bristol, UK).

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Fig. 1.

Chemical structures of BMS-986169 and the phosphate prodrug BMS-986163.

GluN2B Binding K_i Determinations In Vitro.

Studies to determine in vitro binding affinity at rat brain GluN2B receptors were conducted at BBRC. For membrane preparation, rat forebrains were thawed on ice for 20 minutes in homogenization buffer composed of 50 mM KH₂PO₄ (pH adjusted to 7.4 with KOH), 1 mM EDTA, 0.005% Triton X-100, and protease inhibitor cocktail (1:1000; Sigma-Aldrich). Thawed brains were homogenized using a Dounce homogenizer and centrifuged at 48,000g for 20 minutes. The pellet was resuspended in cold buffer and homogenized again using a Dounce homogenizer. Finally, the protein concentration was determined using a BCA kit (Sigma-Aldrich), and the homogenate was aliquoted, flash frozen, and stored at −80°C for 3–4 months. To perform competition binding experiments, thawed membrane was passed through a 24-gauge needle and 98 µl (20 µg) of the membrane protein was added to each well of a 96-well plate. Test compounds (2 µl) were added to achieve the desired concentration in each well of the assay plate, incubated with brain membrane for 15 minutes followed by the addition of [³H]Ro 25-6981 (50 µl, 4 nM final concentration), and further incubation for 1 hour. Nonspecific binding was determined using MK-0657 (40 µM). Membranes were then harvested onto GF/B filter plates using a Filtermate universal harvester (PerkinElmer, Waltham, MA). Prior to membrane transfer, filter plates were presoaked in 0.5% polyethylenimine (PEI) for 1 hour at room temperature and washed three times using cold assay buffer. After membrane transfer, filter plates were washed eight times with cold assay buffer and dried at 50°C for 20 minutes followed by addition of 50 µl of Microscint20 scintillation cocktail (PerkinElmer). Radioactivity was counted using a TopCount (PerkinElmer) after 10 minutes of Microscint addition. Counts per minute were converted to percentage of inhibition and the concentration-response curves plotted and fitted to calculate K_i values using custom-made software. Each experiment had a plate duplicate and was repeated at least twice to obtain an average binding K_i value.

Studies to determine in vitro binding affinity at human (Analytical Biologic Services Inc., Wilmington, DE) and cynomolgus monkey GluN2B receptors were conducted at BMS. Frontal cortical membranes were prepared from frozen brain sections by homogenizing samples at 4°C in hypotonic lysis buffer consisting of 10 mM Tris (pH 7.4), 5 mM EDTA, and protease inhibitors and centrifugation at 32,000g for 20 minutes. The pellet was washed once in buffer consisting of 50 mM Tris (pH 7.4), 1 mM EDTA, and protease inhibitors and centrifuged at 32,000g for 20 minutes. This pellet was then resuspended in buffer containing 50 mM KH₂PO₄ (pH 7.4 at 25°C), 1 mM EDTA, 0.005% Triton X-100, and 0.1% (v/v) Sigma Protease Inhibitor Cocktail. Aliquots were then frozen on dry ice/ethanol and kept at −80°C. On the day of the assay, frozen aliquots of membrane homogenate were thawed, homogenized, and resuspended to provide 15 µg/well cynomolgus brain protein or 35 µg/well human brain protein in assay buffer (5 mM Tris-HCl, pH 7.4). In saturation binding experiments, the membrane preparation was incubated at room temperature for 120 minutes in the presence of increasing concentrations of [³H]Ro 25-6981. Nonspecific binding was defined with 10 µM Ro-04-5595. Competition binding experiments were performed using a single concentration of [³H]Ro 25-6981 (1.5 nM) in the presence of 10 increasing concentrations (in duplicate) of test compound. The reaction was terminated by the addition of 5 ml of ice-cold assay buffer and rapid vacuum filtration through a Brandel Cell Harvester (Brandel, Gaithersburg, MD) using Whatman GF/B filters (PerkinElmer) presoaked in 0.5% PEI. The filtration and washing was completed in less than 30 seconds. The filter was then punched onto a 96-well microbeta sample plate, 200 µl/well of Packard Ultima Gold XR scintillation fluid (PerkinElmer) was added, and the filters were soaked overnight. The filter pads were then counted in an LKB Trilux liquid scintillation counter (PerkinElmer). K_i values from inhibition curves were determined using the “sigmoidal dose-response (variable slope)” non-linear curve fit by GraphPad Prism (version 5.01; GraphPad Software, La Jolla, CA).

Functional Inhibition of NMDA Receptor Subtypes In Vitro.

Studies to demonstrate functional inhibition of GluN2B receptor–mediated currents in Xenopus oocytes were conducted at BBRC. Prior to surgery, frogs were anesthetized using 3-amino-benzoic acid ethyl ester (1 g/l, pH 7.0 adjusted using sodium bicarbonate), the ovarian lobes were removed using aseptic surgical techniques, and the animals were then singly housed for a 24-hour recovery period with postoperative monitoring for up to a week postsurgery. Following removal, the ovarian lobes were incubated in cold Xenopus oocyte buffer A (Biopredic International, Saint Gregoire, France) for 1 hour. The oocytes were then mechanically isolated in small clusters followed by collagenase treatment (1.5–2 mg/ml) for at least 1 hour on a shaking platform at 18°C to remove the follicular layer. The defolliculated oocytes were treated with Xenopus oocyte buffer A, B, and C (Biopredic International) for 15 minutes each in series at 18°C and then maintained in Barth’s solution (pH 7.4) composed of 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO₃, 10 mM HEPES, 0.82 mM MgSO₄, 0.33 mM Ca(NO₃)₂, 0.91 mM CaCl₂, and supplemented with gentamycin (100 µg/ml), penicillin (10 µg/ml), and streptomycin (10 µg/ml). To determine functional inhibition of GluN2B receptors, oocytes were injected with 8–15 ng each of human GluN1a and GluN2B cRNA within 24 hours of isolation. Two electrode voltage clamp recordings were made 2–7 days postinjection. Oocytes were placed in a plexiglass chamber and impaled with glass microelectrodes filled with 3 M KCl. The oocytes were clamped at −40 mV and perfused with buffer containing 90 mM NaCl, 1 mM KCl, 10 mM HEPES, 0.01 mM EDTA, and 0.5 mM BaCl₂, pH 7.4. NMDA receptor currents were activated by the application of 50 µM glutamate and 30 µM glycine and recorded using an Axoclamp 900A amplifier and pClamp 10 data acquisition software (Molecular Devices, LLC., Sunnyvale, CA). The concentration response was determined using four to seven concentrations of the test compound, and each concentration was applied for 15–20 minutes on a different oocyte. The baseline leak current at −40 mV was recorded at the beginning and the end of the recording, and the full current trace was linearly corrected for any change in leak current. The level of inhibition was expressed as the percentage of the initial glutamate/glycine response in the absence of compound, and the IC₅₀ values were obtained by concentration-response curve fitting using the following equation in GraphPad Prism (version 5.01): Y = bottom + (top-bottom)/[1 + 10^(LogIC₅₀ − X) * HillSlope)], where X = log of concentration, Y = percentage of inhibition, and the top and bottom were constrained to 100 and 0, respectively. To determine functional inhibition at other NMDA receptor subtypes, isolated oocytes were injected with 0.2–0.5 ng of human GluN1a and 0.5–1 ng of human GluN2A or 45–60 ng of human GluN1a and 35–55 ng of human GluN2C or GluN2D. The voltage clamp recording procedure was as described earlier except that oocytes were clamped at −80 mV to record GluN2C or GluN2D receptor currents, and compound was applied only at 3 μM for 10-15 minutes. The level of inhibition at 3 µM was calculated as a percentage of the initial glutamate/glycine-mediated current.

Ex Vivo GluN2B Occupancy in Mice and Rats.

Studies to determine ex vivo GluN2B occupancy in rats were conducted at BMS. For dose-response studies, rats (n = 4–5/group) were randomly assigned to receive an i.v. injection via the tail vein of vehicle (0.9% saline, pH 7.4 for BMS-986163 or 30% hydroxypropyl-β-cyclodextrin/70% citrate buffer, pH 4 for BMS-986169; 2 ml/kg), BMS-986169 (0.03, 0.3, 1, or 3 mg/kg), or BMS-986163 (0.1, 0.3, 1, 3, or 10 mg/kg) 15 minutes prior to decapitation, and blood and brains were collected. In a separate study, the time course of GluN2B occupancy was determined from 5 to 120 minutes after i.v. dosing with 3 mg/kg BMS-986169 (n = 4/group). In addition to collection of plasma and brain, a terminal cerebrospinal fluid (CSF) sample was also collected from the cisterna magna for measurement of drug concentrations. Finally, in parallel with [³H]MK-801 binding studies, satellite groups of rats with jugular vein catheters were randomly assigned to receive i.v. BMS-986163 or vehicle treatment (n = 3/group) to determine GluN2B occupancy and drug exposure. Following collection, the brain was placed on ice, the cerebellum was removed, and the forebrain was dissected along the midline into left and right hemispheres, snap frozen in isopentane on dry ice, and stored at −80°C. Blood samples were centrifuged at 3500 rpm for 5 minutes at 4°C to separate the plasma, and plasma and CSF samples were stored at −80°C. On the day of occupancy determinations, one forebrain hemisphere from each animal was thawed and homogenized in 7 volumes of an assay buffer containing 50 mM KH₂PO₄, 1 mM EDTA, 0.005% Triton X-100, and 1:1000 dilution of Sigma protease inhibitor P3843 (pH 7.4) using a Polytron homogenizer (Fisher Scientific, Hampton, NH). In a 96-well plate, 100 µg of tissue (0.4 mg/ml; tested in triplicate) was incubated with 5 nM [³H]Ro 25-6981 in the assay solution at 4°C for 5 minutes. The nonspecific binding was defined by inclusion of 10 µM Ro 25-6981. At the end of the incubation, the reaction was stopped by filtration through FPXLR-196 filters (Fisher Scientific) that had been soaked in 0.5%–1.0% PEI for 1 hour at 4°C. The filters were washed twice with ice-cold assay solution, and the radioactivity was measured using a Wallac Microbeta liquid scintillation counter (PerkinElmer). For each sample, specific binding was calculated by subtracting the value of the nonspecific binding from that of the average total binding and percentage of occupancy calculated as (1 − specific binding in drug treated/specific binding in vehicle treated) × 100%. For estimation of the dose producing 50% GluN2B occupancy (Occ50), a one-site binding model using nonlinear regression was fitted to the mean occupancy achieved at each dose (GraphPad Prism version 7.02). To estimate the Occ50 drug concentration in plasma, brain, or CSF, the same curve fit was applied to plots of occupancy/exposure results for individual subjects.

Additional GluN2B occupancy determinations were also conducted at BBRC in mice completing behavioral assessment in the FST (n = 4/group) or in satellite groups dosed in parallel with NSF and LMA subjects (n = 4/group). Mice were rapidly decapitated, and brain and blood samples were collected. Forebrain membranes were prepared as described earlier except for the addition of 3 ml of assay buffer for 160 mg of tissue and homogenization by the Polytron for 10 seconds followed by 30 strokes using a Dounce homogenizer. On the day of the experiment, brain homogenates were thawed on ice and passed three times through a 24-gauge needle. In a 96-well plate, 200 µl (6.4 mg/ml) of tissue homogenate (tested in triplicate) was incubated with 6 nM [³H]Ro 25-6981 in assay buffer at 4°C for 5 minutes on a shaking platform. The membrane was harvested onto a GF/B filter plate (treated with 0.5% PEI for 1 hour at 4°C), then the filter plate was dried at 50°C for 20 minutes and 50 µl of Microscint20 (PerkinElmer) was added. After shaking of the filter plate for 10 minutes, the radioactivity was measured using a TopCount (Perkin Elmer). Nonspecific binding was determined by incubating the membrane from vehicle-treated animals with 10 µM Ro 25-6981.

In Vivo [³H]MK-801 Binding in Rats.

Studies to determine inhibition of in vivo [³H]MK-801 binding were conducted at BMS using methods previously described (Fernandes et al., 2015). For the dose-response study, rats with previously implanted jugular vein catheters were randomly assigned (n = 4/group) to treatment with either vehicle (0.9% saline, 2 ml/kg, i.v.), BMS-986163 (0.1, 0.3, 1, 3, 10, or 30 mg/kg, i.v.), or MK-801 (5 mg/kg, i.v.) to define the specific binding window. All rats received [³H]MK-801 (0.2 µCi/g body weight) 5 minutes later and were decapitated 10 minutes after [³H]MK-801 administration (i.e., 15 minutes after BMS-986163 treatment). For the time-course study, rats (n = 4/group) were dosed with BMS-986163 (3 mg/kg, i.v.) 5–120 minutes prior to euthanasia, with all subjects receiving [³H]MK-801 10 minutes prior to sacrifice. At the appropriate time point, the brain was collected, the cerebellum and brain stem were removed, and one forebrain hemisphere was weighed and homogenized in 30 volumes of ice-cold 5 mM Tris-acetate buffer (pH 7.0). A 600-µl sample of the homogenate was filtered through GF/B Whatman filters (presoaked in ice-cold Tris-acetate buffer) on a filter box connected to a vacuum source. Filters were washed four times with ice-cold Tris-acetate buffer, and then placed in a scintillation vial with 10 ml of scintillation fluid (Optiphase supermix; PerkinElmer) overnight. Radioactivity in the scintillation vial was counted using a Wallac Microbeta liquid scintillation counter. The total [³H]MK-801 binding was determined by measuring radioactivity in the forebrain of animals treated with vehicle, whereas nonspecific binding was determined by measuring the radioactivity in animals treated with MK-801. For each sample, specific binding was calculated by subtracting the value of the nonspecific binding from that of the total binding, and percentage of inhibition was calculated as (1 − specific binding in drug treated/specific binding in vehicle treated) × 100%. For estimation of the dose producing 50% inhibition of MK-801 binding, data were fitted to a one-site binding model using nonlinear regression as described previously.

Mouse FST.

Studies to evaluate GluN2B NAM treatment effects on immobility in the mouse FST were conducted at BBRC. All studies were conducted between 09:00 and 13:30 hours and were performed under dim light and low noise conditions. Behavior was recorded and quantified using the automated CleverSys forced swim test apparatus (CleverSys, Reston, VA), and investigators were unblinded to dosing solutions. Animals (n = 8–11/group) were randomly assigned to treatment with either vehicle (30% hydroxypropyl-β-cyclodextrin/70% citrate buffer, pH 4; 5 ml/kg, i.v.), CP-101,606 (3 mg/kg, i.v.), or BMS-986169 (0.3, 0.56, 1, or 3 mg/kg, i.v.) and 15 minutes later were placed in plexiglass swim tanks (20-cm diameter, 40-cm height) filled with water up to a height of 20 cm at a temperature of 25 ± 1°C. In a separate study, mice were randomly assigned to treatment (n = 9–10/group) with either vehicle (0.9% saline; 10 ml/kg, i.v.) or ketamine (1, 3, or 10 mg/kg, i.v.) and tested 30 minutes after dosing. Swim tanks were positioned inside a box made of plastic and separated from each other by opaque plastic sheets to the height of cylinders. Individual animals were placed in one of the three swim tanks, and behavior was recorded in a 6-minute testing session. The total immobility duration, defined as the time spent passively floating with only small movements necessary to keep the nose/head above water and remain afloat during the 6-minute test, was measured using the automated CleverSys software. Results were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test (GraphPad Prism version 7.02). Upon completion of testing (∼25 minutes after drug treatment), a subset of animals (n = 4/group) were euthanized by rapid decapitation, and blood and brain samples were collected for GluN2B occupancy and drug exposure measurements as previously described.

NSF in Mice.

Studies to examine the effect of GluN2B NAM treatment on NSF were conducted at BBRC. Prior to treatment, all mice were briefly (∼1 minute) placed in a restrainer to acclimatize them to the i.v. dosing conditions for 2 consecutive days. Animals were then randomly assigned to treatment (n = 12–15/group) with CP-101,606 (10 mg/kg, i.v.), BMS-986163 (1, 3, or 10 mg/kg, i.v.), or their respective vehicles (25% hydroxypropyl-β-cyclodextrin or phosphate buffer, pH 7.4, respectively; 5 ml/kg, i.v.) and then food deprived for 21 hours with water available ad libitum. On the following day (i.e., 24 hours after treatment), individual mice were placed in a novel Perspex arena (40 × 40 × 40 cm; Coulbourn Instruments, Allentown, PA) covered on three sides with white paper, with the fourth side left transparent to enable behavioral evaluation. A light source was positioned on the top to produce a light intensity of ∼1000 lux in the center of the arena. One food pellet (standard chow feed) was placed on the top of a 60-mm Petri plate placed in the center of the arena. The session began with the animal placed in one corner of the arena facing away from the food pellet. The latency to begin eating the pellet (i.e., latency to feed) was recorded by an observer blinded to drug treatment. Feeding was defined as the mouse holding the food pellet with its forepaws and biting it, and testing was terminated either when the mouse started to feed or after 6 minutes had elapsed. After evaluation in the novel arena, the mouse was transferred to its home cage and food consumption was monitored for 30 minutes (home cage feeding). Results were analyzed by either two-tailed, unpaired t test (CP-101,606 vs. vehicle) or one-way ANOVA followed by Dunnett’s post-hoc test (GraphPad Prism version 7.02). Satellite groups of mice (n = 4/treatment) were dosed in parallel with the NSF subjects, and plasma and brain samples were collected 15 minutes later for exposure and GluN2B occupancy determinations.

During the initial pharmacological characterization of the NSF assay, studies were conducted to examine the effect of ketamine treatment following i.p. administration, a dosing route commonly used to examine the antidepressant-like effect of this agent. Subjects (n = 12–15/group) were treated with vehicle (Milli-Q water, 10 ml/kg) or ketamine (3, 10, or 30 mg/kg) and tested in the NSF assay 24 hours later as described earlier.

Ex Vivo Hippocampal LTP in Rats.

Studies to examine the effects of GluN2B NAM treatment on hippocampal LTP were conducted by BMS. Rats were handled and acclimatized to the i.v. dosing restrainer for up to 5 minutes each day for 3 days prior to dosing. On the morning of drug treatment, rats were restrained and dosed via the tail vein by an investigator unblinded to treatment with either BMS-986169 (1 or 3 mg/kg) or vehicle (30% hydroxypropyl-β-cyclodextrin/70% citrate buffer; 2 ml/kg). Typically, either two rats (one treated, one control) or four rats (two treated, two control) were randomly assigned to treatment each day for subsequent LTP measurements 24 or 72 hours later. Each dose of BMS-986169 or time point was examined as a separate study with independent vehicle-treated controls (n = 5–8/group). At the appropriate time point, rats were rapidly decapitated, and brain tissue was removed and quickly submerged into ice-cold cutting solution containing 100 mM sucrose, 60 mM NaCl, 3 mM KCl, 7 mM MgCl_2•6H₂O, 1.25 mM NaH₂PO₄, 0.5 mM CaCl₂, 5 mM d-glucose, 0.6 mM ascorbate, and 28 mM NaHCO₃. The cerebellum and frontal cortex were removed, and the brain was cut down the midline. Transverse slices (350 µm) were cut from the middle of the hippocampal formation with a vibratome (Leica VT1000S; Leica Microsystems, Wetzlar, Germany) in ice-cold cutting solution bubbled with 95% O₂/5% CO₂. Brain slices were transferred to a 50:50 mixture of cutting solution and artificial cerebrospinal fluid (aCSF) containing 124 mM NaCl, 3 mM KCl, 1 mM MgSO_4•7H₂O, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 10 mM d-glucose, and 36 mM NaHCO₃ and oxygenated with 95% O₂/5% CO₂ at room temperature (26°C) for 30 minutes. Slices were then switched to 100% aCSF and continued to be oxygenated with 95% O₂/5% CO₂ at 30°C for at least 30 minutes of recovery. Up to eight slices were recorded simultaneously using a modified multislice recording configuration (Graef et al., 2013). In brief, two separate tissue slice chambers, each with a capacity for four isolated tissue slices, were perfused with warm (32–34°C), oxygenated aCSF using a gravity perfusion system. A flow rate of 2 ml/min for each four-slice recording chamber was regulated by maintaining a constant fluid level in a secondary solution chamber through the use of a solenoid valve (Harvard Apparatus, Holliston, MA), a float, and an IR beam detector (NV-253SD; Med Associates, Fairfax, VT). Four vehicle slices were placed in one of the four-slice recording chambers, and four slices from a rat treated with test agent were placed in the other four-slice recording chamber, with the chambers used for each condition being alternated on subsequent experimental days. Once all slices were oriented in the recording chambers, custom-made recording and stimulating electrodes (0.2-µm diameter platinum/iridium wire) were positioned in the stratum radiatum layer of the Cornu Ammonis area 1 (CA1) region of the hippocampus. Field responses were elicited with a single monophasic stimulation from two twisted wires (0.1 ms) every 20 seconds (3 Hz) using individual IsoFlex stimulus isolator units driven by a Master 8 stimulus generator (A.M.P.I.; Jerusalem). The strength of each stimulus was adjusted to elicit 30%–50% of the maximum response. Baseline signals were recorded for 20 minutes, then LTP was induced by the following high-frequency stimulation (HFS) protocol: two trains of 80 stimulations at 100 Hz at the same stimulus intensity with a 60-second interval between trains. This HFS protocol was repeated two more times at 20 and 40 minutes following the first bout of HFS. Following this conditioning stimulus, a 1-hour test period was recorded where responses were again elicited by a single stimulation every 20 seconds (3 Hz) at the same stimulus intensity. Signals were amplified with a differential amplifier and were digitized and sampled at 10 kHz with a DigiData 1440 (Molecular Devices, Sunnyvale, CA), recorded using Clampex 10.0 acquisition software, and analyzed with the Clampfit 10.0 software package (Molecular Devices). Data from all four slices from each animal (control and treated) on each experimental day were averaged to obtain mean LTP values for each subject. Only data from slices where the baseline responses were greater than 0.1 mV and did not vary by more than 10%, and demonstrated at least a 10% potentiation of the baseline response were used for analysis. These inclusion criteria were prespecified prior to the LTP recordings and resulted in average values from two to four slices per subject. The total numbers of slices excluded from analysis across all studies were as follows: LTP amplitude: vehicle = 9/78 (11.5%), BMS-986169 = 9/79 (11.4%); LTP slope: vehicle = 8/78 (10.3%), BMS-986169 = 9/79 (11.4%). The numbers of slices excluded from analysis for individual studies were as follows: study 1: vehicle (24 hours) = 1/27 slices, 3 mg/kg BMS-986169 (24 hours) = 3/27 slices (slope) or 4/27 slices (amplitude); study 2: vehicle (72 hours) = 4/31 slices, 3 mg/kg BMS-986169 (72 hours) = 4/32 slices; study 3: vehicle (24 hours) = 3/20 slices (slope) or 4/20 slices (amplitude), 1 mg/kg BMS-986169 (24 hours) = 2/20 slices (slope) or 1/20 slices (amplitude). One-tailed paired t tests comparing corresponding test periods between control and treated animals were used to test for statistical significance.

Locomotor Activity in Mice.

Studies to examine the effect of GluN2B NAM treatment on mouse locomotor activity were conducted at BBRC and performed using an automated LMA apparatus. All testing was conducted between 09:00 and 13:30 hours in a dark room with low noise conditions, and investigators were unblinded to dosing solutions. Mice were habituated to individual chambers (40 × 40 × 40 cm; Coulbourn Instruments) for 2 hours and then randomly assigned to treatment. In an initial study, the dose response to ketamine was examined in mice (n = 8–9/group) treated with vehicle (Milli-Q water, 5 ml/kg, i.v.) or ketamine (3, 10, 30 mg/kg, i.v.). In the subsequent study, mice (n = 7–8/group) were treated with either vehicle (25% hydroxy-β-cyclodextrin/75% water; 5 ml/kg, i.v.), ketamine (30 mg/kg, i.v.), or BMS-986169 (3, 10, or 30 mg/kg, i.v.). Animals were returned to the chamber, and activity data were acquired every 250 ms from the floor panel sensor and expressed as ambulatory distance (centimeters) recorded in 10-minute time bins using TruScan 2.0 software (Coulbourn Instruments). Results were analyzed by two-way repeated-measures (RM) ANOVA followed by Dunnett’s post-hoc analysis (GraphPad Prism version 7.02). Separate satellite groups (n = 4/treatment) were dosed in parallel, and plasma and brain tissue was collected 15 minutes later to determine drug concentrations and GluN2B occupancy.

Abnormal Behaviors in Cynomolgus Monkeys.

Studies to examine the effect of GluN2B NAM treatment on spontaneous behaviors in monkeys were conducted by BMS. Prior to treatment, baseline behavioral profiles were established by observing home cage behavior for 15 minutes/wk for 3 weeks. Behaviors were evaluated using a 12-item behavioral checklist previously used to demonstrate psychotomimetic-like behaviors following treatment with amphetamine or ketamine in macaques (Castner and Goldman-Rakic 1999; Roberts et al., 2010; Supplemental Table 1). When a given behavior was observed, a mark was made in the appropriate time bin. Each study was organized using a Latin-square design, and the observer was blinded to treatment. A 5-minute predose observation was made 30 minutes before treatment with vehicle (30% hydroxypropyl-β-cyclodextrin/70% water, 0.4 ml/kg, i.m.), (±)ketamine (1 mg/kg, i.m.), or BMS-986169 (1, 3, or 5.6 mg/kg, i.m.), and behavior was evaluated 0–5, 5–10, 10–15, 30–35, and 60–65 minutes post-treatment (n = 8). To examine a higher dose of 10 mg/kg BMS-986169, a second blinded study was conducted using a Latin-square design and a two-injection protocol whereby 2 × 0.4-ml/kg injections were given to achieve a total dose of 10 mg/kg BMS-986169, 1 mg/kg ketamine, or two injections of vehicle (n = 8). The dose of ketamine used for comparison was selected from a separate study showing a dose-dependent increase in behavior after ketamine administration (n = 12). Behaviors were summed over the entire session and expressed in terms of change from preinjection baseline. Composite measures of “all abnormal behaviors” (behaviors 2+4+6+7+10+11; Supplemental Table 1) and “hallucinatory/dissociative” (behaviors 7+11; Supplemental Table 1) were calculated and analyzed by one-way RM ANOVA followed by Dunnett’s post-hoc test (GraphPad Prism version 7.02). On completion of the evaluation, subjects were placed in a primate restraint chair, and a plasma sample (∼75 minutes postdose) was collected from the saphenous vein for measurement of drug concentrations.

Cognitive Impairment in Cynomolgus Monkeys.

Studies to examine the effect of GluN2B NAM treatment on cognitive performance were conducted at BMS using a list delayed match to sample (list-DMS) procedure described previously (Weed et al., 2016). Subjects (n = 9) received prior treatment with other pharmacological agents and were given at least a 2-month drug-free period prior to the start of these studies. During the procedure, animals were seated in restraint chairs and placed within a sound-attenuated chamber containing a touch-sensitive computer monitor controlled by the Monkey CANTAB (Cambridge Neuropsychological Testing Automated Battery) software (Lafayette Instruments, Lafayette, IN). Following correct responses, a dispenser delivered 190-mg banana flavored pellets (Bioserv, Frenchtown, PA). Animals were first trained to proficiency on the standard CANTAB DMS task in which an abstract stimulus is presented in the “sample” phase, and after a memory delay, the sample stimulus plus three distracter stimuli are presented in the “choice” phase. Animals then progressed to a list-DMS procedure in which three different sample stimuli are presented first followed by their choice phases which occur sequentially based on their respective memory delays. Thus, the three trials are nested within the longest memory delay, thereby reducing the overall session length. The memory delays used in this study were 2, 20, and 45 seconds. Completion of the list was followed by a 10-second screen blank, after which a new list was presented for a total of 20 lists in each test session. During both the sample and choice phases, the subject had 5 seconds to respond to the stimulus, otherwise the trial was considered an omission. If the omission was in the sample phase, the choice phase for that sample stimulus was not presented, but the timing of the sample and choice phases for other stimuli remained unchanged. All subjects were habituated to receiving injections prior to testing. Evaluation of performance after vehicle treatment (25% hydroxypropyl-β-cyclodextrin/75% water, 0.4 ml/kg, i.m.) occurred prior to, during, and at the end of the dose-response function (total of four vehicle determinations). The effect of BMS-986169 treatment was tested twice weekly (Monday and Thursday) with baseline performance sessions on Tuesday and Friday. On test days, the cohort was divided roughly in half, and one half received a given dose of BMS-986169 and the other half received a different dose. Drug doses were thus administered in a mixed order across test days and across subjects by investigators unblinded to treatment. In the first study, the effect of BMS-986169 (0.1, 0.3, 1, or 3 mg/kg, i.m.) administered 30 minutes prior to testing was examined to determine the dose-effect relationship. In a follow-up study, vehicle or 1 mg/kg BMS-986169 was administered 3 or 5 hours prior to testing to examine the time course of the effect. List-DMS test sessions were ∼30 minutes long, and on completion, blood samples were collected from the saphenous vein for analysis of plasma drug concentrations. Sample collection times, therefore, correspond to 1 hour postdose for the dose-response study and 3.5 or 5.5 hours for the time-course study. Plasma and CSF drug concentrations were also investigated after dosing with BMS-986169 (0.3–5.6 mg/kg, i.m.) in a separate cohort of cynomolgus monkeys previously implanted with vascular and CSF lumbar access ports (n = 2/group). Blood samples were collected into K₂EDTA tubes on ice, plasma was separated by centrifugation at 1330g for 10 minutes, and plasma and CSF samples were stored at −80°C until analyzed.

The primary measure for these studies was performance accuracy expressed as the percentage of correct trials (% accuracy = [correct trials]/[(correct trials + errors)] × 100). Secondary measures included 1) latency to respond in the choice phase (milliseconds between onset of stimulus presentation and touching of monitor) and 2) percentage of task completed (trials with a response in both sample and choice phase/trials presented). Omissions were not included in the percentage correct measure, but contributed to percentage of task completed. Percentage correct accuracy was analyzed by two-way RM ANOVA followed by Dunnett’s post-hoc test. For the time-course study, performance after vehicle treatment was similar regardless of the pretreatment time (0.5, 3, or 5 hours), and the average was therefore used in the analysis. Latencies to respond in the choice phase (averaged across delays) and percentage of task completed for the entire session were analyzed by one-way RM ANOVA followed by Dunnett’s post-hoc test. In addition, for the long delay condition, the difference between performance after vehicle and drug treatment was calculated for each subject as follows: difference = % correct at long delay(drug treatment) − % correct at long delay(vehicle treatment). All statistical analyses were conducted using GraphPad Prism (version 7.02).

qEEG Studies in Cynomolgus Monkeys.

Studies to examine the effect of GluN2B NAM treatment on the qEEG response were conducted by BMS using methods described previously (Keavy et al., 2016). Six cynomolgus monkeys served as subjects for the qEEG studies, and each had been used previously in pharmacological studies, with a drug-free period of at least 1 month prior to testing. Subjects were surgically implanted under isoflurane anesthesia with radiotelemetry transmitters (Konigsberg Instruments, Inc., Monrovia, CA) attached to two sets of EEG leads placed over the dura: one set with leads over the frontal cortex (with a reference over parietal cortex; roughly F6-P6 in the 10–20 system), and the second set with leads over the auditory cortex (roughly C6-CP6 in the 10–20 system). Buprenorphine (0.01–0.03 mg/kg, i.m.) was administered after surgery and for a further 2–3 days twice daily with extended treatment as needed if subjects showed signs of discomfort. After full recovery (approximately 3–4 weeks), the implanted radiotelemetry device was tested to ensure a good EEG signal, and suitable subjects were enrolled in the study. On each test day, prior to the start of qEEG recordings, subjects were seated in a primate restraint chair and an i.v. catheter was placed in a saphenous vein for drug administration. Animals were allowed to acclimate to a quiet testing chamber outfitted with an antenna to receive telemeter signals and a camera to monitor the animal during the session. After a 20-minute baseline recording, animals were treated with either vehicle (0.9% saline; 0.4 ml/kg, i.v.) or BMS-986163 (0.12, 0.36, or 1.2 mg/kg, i.v.) by investigators unblinded to treatment, and the qEEG was measured for a further 90 minutes. The i.v. catheter was removed at the end of the session, and animals were tested once weekly with treatments assigned using a Latin square design. Analog signals from the telemeters were digitized at 1000 Hz by PowerLab DAQ systems (ADInstruments, Colorado Springs, CO). Raw EEG waveforms were Fourier transformed using a Han (cosine bell) window with 50% overlap between data blocks and an FFT block size of 1024. Overall total power (0.5–55 Hz) as well as power within the different frequency bands was recorded for delta (0.5–4 Hz), theta (4–9 Hz), alpha (9–13 Hz), beta 1 (13–19 Hz), beta 2 (20–30 Hz) and gamma (30–55 Hz) bandwidths. The relative power (power in a band/overall total power) was determined as the average value from 1-minute bins across the 90-minute session and analyzed by two-way RM ANOVA followed by Holm-Sidak post-hoc test (GraphPad Prism version 7.02). Area under the curve over the entire 90-minute period for each power band was calculated from the time-course data and analyzed by RM ANOVA followed by Dunnett’s post-hoc test (GraphPad Prism version 7.02). Finally, using the area under the curve results, Cohen’s d estimate of effect size was calculated using unpaired statistics (http://www.uccs.edu/∼lbecker/).

Plasma and CSF drug concentrations were also investigated after dosing with BMS-986163 (1.2 mg/kg, i.v.) in cynomolgus monkeys previously implanted with vascular and CSF lumbar access ports (n = 2/time point). Sample collection, processing, and storage were as previously described.

Quantification of BMS-986169 and BMS-986163 in Samples from In Vivo Studies.

For exposure analysis of BMS-986169 and BMS-986163 in plasma, brain, and CSF, rats and monkeys were dosed and samples were collected as described. BMS-986163 was stabilized ex vivo by collecting blood samples into tubes containing 4% K₂EDTA dissolved in 1 M phosphate buffer, pH 7.4 (5% of the blood volume) and maintained at 4°C. Plasma was gathered within 15 minutes of blood collection and stored at −80°C until analysis. For brain samples, tissue was homogenized and diluted with blank plasma. CSF samples were prepared in the same manner as plasma. An aliquot of 50 µl of calibration standard or study sample (plasma/tissue/CSF) was added to 200 µl of acetonitrile containing internal standard (Ro 25-6981 at 100 nM) and transferred to a Strata protein precipitation filter plate (Phenomenex, Torrance, CA). Plates were vortexed on a plate shaker for 5 minutes at 300 rpm, centrifuged at 4000 rpm for 5 minutes, and the eluent was collected into a 96-well collection plate (Waters Corporation, Milford, MA). A volume of 4 µl was injected for liquid chromatography–mass spectrometry/mass spectrometry analysis. Liquid chromatography–mass spectrometry/mass spectrometry analysis was performed on a Waters Acquity UPLC (Waters Corporation) interfaced to an API4000 triple quadrupole mass spectrometer (Sciex, Toronto, Canada) equipped with a Turboionspray source. The source temperature was set at 550°C and the ionspray voltage was set to 4.5 kV. Samples were maintained at 10°C for the duration of the analysis. Separations were performed on a Waters Acquity BEH C18 (2.1 × 50 mm, 1.7 µm) column maintained at 60°C. The mobile phase, which consisted of 0.1% (v/v) formic acid in water (A) and 0.1% (v/v) formic acid in acetonitrile (B), was delivered at a flow rate of 700 µl/min. Separation was achieved using a gradient elution starting at 95% aqueous mobile phase and going to 95% organic mobile phase. Nitrogen was used as nebulizer and auxiliary gas with a pressure and flow rate of 80 psi and 7 l/min, respectively. Data acquisition used selected reaction monitoring operating mode. Standard curves used for quantification were fitted with linear regression weighted by reciprocal concentration, 1/x². Data and chromatographic peaks were quantified using Analyst version 1.4.2 software (Sciex).