Drugs and Reagents.

JNJ-42226314 was synthesized at Janssen Research & Development, LLC (Fig. 1). Synthesis is described in U.S. patent 8,362,000. The tool compound SAR-127303 (1,1,1,3,3,3-hexafluoropropan-2-yl 4-(((4-chlorophenyl)sulfonamido)methyl)piperidine-1-carboxylate) was also synthesized at Janssen Research & Development, LLC according to procedures described in the literature (Griebel et al., 2015). 1,1,1,3,3,3-Hexafluoropropan-2-yl 4-(((4-chlorophenyl)sulfonamido-2-tritio)methyl)piperidine-1-carboxylate ([³H] SAR-127303) (see Supplemental Material) and 1,3-dihydroxypropan-2-yl-1-tritio oleate ([³H] 2-OG) were prepared at Moravek, Inc. (Brea, CA). [³H] 2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxypropyl)cyclohexyl]-5-(2-methyloctan-2-yl)phenol (CP-55940) was purchased from Perkin-Elmer (Waltham, MA). N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-carbamic acid 4′-(aminocarbonyl)[1,1′-biphenyl]-4-yl ester (WWL-70), 4-nitrophenyl-4-[bis(1,3-benzodioxol-5-yl)(hydroxy)methyl]piperidine-1-carboxylate (JZL-184), 4-[bis(1,3-benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 2,2,2-trifluoro-1-(trifluoromethyl)ethyl ester (KML-29), and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (AM-251) were obtained from Tocris (Minneapolis, MN). N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide (PF-3845) was purchased from Sigma-Aldrich (St. Louis, MO). Methyl arachidonyl fluorophosphonate (MAFP) was obtained from Cayman Chemical (Ann Arbor, MI). The fluorophosphonate carboxytetramethylrhodamine (FP-Rh) (Liu et al., 1999) probe was synthesized according to previously reported procedures (Kidd et al., 2001; Patricelli et al., 2001) and modifications thereof. The synthesized material was purified by preparative high-performance liquid chromatography (HPLC) (Column: Xtimate C18 150*25 mm*5 μm) under mildly basic conditions (A: 10 mM ammonium bicarbonate, B: acetonitrile). After lyophilization, pure FP-Rh was obtained as a purple solid that was stored under inert conditions at −20°C until further use or at −80°C as argon-purged DMSO aliquots. Structure and analytical purity were confirmed by NMR (¹H, ³¹P, ¹⁹F) and electrospray ionization mass spectrometry (ESI-MS) techniques.

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Fig. 1.

Chemical structure of JNJ-42226314, [1-(4-fluorophenyl)indol-5-yl]-[3-[4-(thiazole-2-carbonyl)piperazin-1-yl]azetidin-1-yl]methanone.

Compounds were dissolved in DMSO for in vitro studies. For in vivo studies, drugs were freshly prepared daily in 20% (2-hydroxyproplyl)-β-cyclodextrin (HP-β-CD) for intraperitoneal administration.

In Vitro MAGL Activity Assay.

The assay used to measure the in vitro activity of MAGL was adapted from the substrate cleavage assay used for another serine hydrolase, fatty acid amide hydrolase (FAAH), described in Wilson et al. (2003). The assay consists of combining MAGL from HeLa cells, rat/mouse brain membranes, or human peripheral blood mononuclear cells with test compounds (JNJ-42226314, JZL-184, KML-29, and SAR-127303); adding [³H] 2-OG; and measuring the amount of cleaved [1,3-³H]-glycerol that passes through an activated carbon filter. The amount of cleaved [³H]-glycerol passing through the carbon filter is proportional to the activity of the MAGL enzyme. The substrate [³H] 2-OG (Moravek) was found to be more stable than [³H] 2-AG (Moravek) and, therefore, was used for this assay.

HeLa cells (ATCC, Manassa, VA) were grown to confluence in 15-cm tissue culture dishes in complete medium (Eagle’s minimum essential medium; Lonza, Basel, Switzerland) containing 10% fetal bovine serum, nonessential amino acids, sodium pyruvate, glutamine, and 100 IU/ml penicillin/streptomycin. Once the cells reached confluence, the medium was aspirated, and cells were removed with Dulbecco’s phosphate-buffered saline and 5 mM EDTA. The cells were counted and then centrifuged for 5 minutes at 300g, and the supernatant was removed. The HeLa cell pellet was homogenized using a Polytron in assay buffer containing 125 mM Tris, 1 mM EDTA, 0.02% Triton-X100, and 0.4 mM HEPES (pH = 7.4) in a volume yielding 500,000 cells/ml. The enzyme reaction combined test compound (0.1 nM to 10 μM), HeLa cell homogenate (diluted to a final concentration of 2000 cell equivalents/well final), and 300 nM [³H] 2-OG (70,000 counts per minute (CPM)/well, specific activity 10–20 Ci/mmol). Compounds (from 0.1 to 10 μM, diluted from 10 mM DMSO stocks) were incubated with the HeLa cell homogenate for 30 minutes prior to adding the radiolabeled substrate, and then the complete mixture was incubated for 1 hour at room temperature in a total volume of 25 μl/well. In preparation for the filtration step, powdered charcoal was suspended in methanol and vortexed to wet the charcoal. Glycerol was added to help maintain the suspension, and the solution was buffered to pH 7.0 with 100 mM Tris. The final suspension was 20% v/v charcoal, 20% v/v methanol, and 20% v/v glycerol in 100 mM Tris. Thirty-five microliters of the charcoal mix was added per well, and the liquid was vacuumed through the filter plate prior to adding the reaction sample. After enzyme incubation for 1 hour at room temperature, 20 μl of the reaction was transferred to the charcoal plate and incubated for 5 minutes. The filter plate was then placed on top of a recipient plate containing 65 μl Microscint-PS. The reaction was centrifuged to flow the reaction through the charcoal and into the Microscint-PS. The plate containing the Microscint-PS and reaction product was then counted on a Top Count the next day. Percentage inhibition of enzyme activity was calculated using the following formula: % inhibition = 100 − ((CPM observed – low control CPM)/(high control CPM – low control CPM) × 100), where low control CPM = substrate + HeLa cell homogenate + 10 μM MAGL inhibitor and high control CPM = substrate + HeLa cell homogenate. IC₅₀ values were calculated using a one-site model with GraphPad Prism 7.0 (San Diego, CA).

Testing of compound potency was also performed in human peripheral blood mononuclear cells (PBMCs). To harvest PBMCs, 1 ml of fresh whole human blood was aliquoted into a 14-ml Falcon tube (Becton Dickinson, Franklin Lakes, NJ) with 4 ml of room temperature Red Blood Cell Lysing Buffer (Sigma). Tubes were set to incubate at room temperature for 30 minutes using a rocking platform to gently invert the sample. After complete lysis of the sample, tubes were centrifuged at 1800g for 10 minutes at room temperature. The supernatant containing lysed red blood cell membranes was carefully removed from the white blood cell pellet, which was resuspended in 1 ml Red Blood Cell Lysing Buffer and centrifuged at 1800g for 10 minutes at room temperature. The supernatant was removed, and the pelleted PBMCs were frozen at −80°C until use. For the activity assay, the thawed pellet was resuspended in 50 ml assay buffer and used in the enzyme reaction as previously described.

Testing of compound potency was also performed in mouse and rat soluble brain fractions. Fresh-frozen mouse or rat brain was homogenized by Polytron in 10 ml/g Triton-free 125 mM Tris, 1 mM EDTA, and 0.4 mM HEPES (pH = 7.4) on ice. The homogenate was centrifuged at 1000g for 10 minutes at 4°C. The supernatant fraction was then centrifuged at 35,000g for 30 minutes at 4°C. The supernatant fraction from the high-speed centrifugation was kept on ice and used as the enzyme source for the reaction. The supernatant fraction was diluted ∼200× in assay buffer to yield an approximate final protein concentration of 17 μg/ml and used in the enzyme reaction as previously described.

Expression and Purification of Recombinant Human MAGL.

Recombinant human MAGL was prepared according to a procedure published previously (Labar et al., 2007). Briefly, N-terminal 6-His–tagged and C-terminal strep–tagged human MAGL was overexpressed in Escherichia coli BL21(DE3) cells. After cell pellet lysis, MAGL was purified from crude extract by nickel, size exclusion, and ion exchange (Hi trap Q) chromatography. Protein purity was evaluated by SDS-PAGE electrophoresis. Pure fractions were pooled, concentrated, and stored at −80°C in 50 mM sodium carbonate containing 208 mM NaCl and 1 mM Tris(2-carboxyethyl)phosphine (pH = 9.5).

Inhibition Kinetics.

The kinetic mechanism of inhibition of human recombinant MAGL by JNJ-42226314 was determined via an enzymatic assay quantifying arachidonic acid by RapidFire-MS. Briefly, responses of JNJ-42226314 were tested at various substrate (2-AG) concentrations, with initial rates (υ) analyzed as a function of substrate (S) and inhibitor (I) concentrations through a global fit via least-squares method to determine the inhibition mechanism according to eq. 1 below (Copeland, 2005). V_max in these equations refers to the maximum velocity, K_m the Michalis-Menten constant, K_i the inhibition constant for the formation of the binary enzyme inhibitor complex, and α the substrate and inhibitor interaction constant for the formation of the tertiary enzyme-substrate inhibitor complex. All enzymatic reactions were run in 50 mM Tris-HCl containing 50 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 0.002% Tween-20, 0.01 mg/ml bovine serum albumin (BSA), 7.5% DMSO, and 2.5% ethanol (pH = 7.5) in a total volume of 40.4 μl. Inhibitor (0.4 μl), which was serially diluted in pure DMSO, was added to a 384-well plate (Greiner, Monroe, NC). Substrate (2-AG) (20 μl), which was serially diluted in assay buffer, was added to the plate containing the test inhibitor. Human recombinant MAGL (20 μl, final concentration = 1 nM) was added to the substrate-inhibitor mixtures to initiate the reaction. The reaction mixtures were incubated at room temperature (22°C) for 10 minutes, and the reactions were quenched by adding 10 µl formic acid to a final concentration of 1%. The amount of arachidonic acid produced was quantified by RapidFire-MS in a mobile phase of 0.1% acetic acid in water (solution A) and 90% acetonitrile in 0.1% acetic acid (solution B).(1)

In Vitro Equilibrium Binding Assay Using [³H] SAR-127303.

Compound binding to MAGL was evaluated in plasma membrane preparations from rat, mouse, and human brain samples using [³H] SAR-127303 (Moravek). Whole rodent brains were collected immediately after sacrifice by CO₂ inhalation and frozen on dry ice. The human brain sample, a fragment of frontal cortex, was obtained from the Anatomy Gifts Registry (Hanover, MD). The subject was an 84-year-old male who perished of causes not directly attributable to CNS disease. Brain samples from all species were homogenized using a PowerGen 35 homogenizer (Fisher Scientific, Hampton, NH) in 10 ml/g 125 mM Tris, 1 mM EDTA, and 0.4 mM HEPES (pH = 7.5) on wet ice. Membrane fractions were prepared by centrifugation. First, debris was removed by low-speed centrifugation (1000g for 10 minutes at 4°C). The supernatant fraction from the first centrifugation was then centrifuged at 35,000g for 30 minutes at 4°C. The supernatant fraction from the second centrifugation was discarded, and the membrane pellet was reconstituted in 125 mM Tris, 1 mM EDTA, and 0.4 mM HEPES (pH = 7.5). Total membrane protein was estimated using a Nanovue (GE Healthcare Life Sciences, Pittsburgh, PA). Membrane samples reconstituted from the membrane pellet were stored at −80°C and used after one thaw.

For equilibrium binding assays, membrane homogenate (350 μg total membrane protein per reaction) was combined with radioligand at various concentrations of the unlabeled compound of interest in a total volume of 100 μl. The radioligand was [³H] SAR-127303 (Moravek) prepared at a specific activity of 10 Ci/mmol and used at a final concentration of 8 nM. Compounds were diluted from 10 mM DMSO stocks. All components were diluted in assay buffer containing 10 mM Tris, 1 mM EDTA, and 1% bovine serum albumin (pH = 7.0). Reactions were incubated at 21°C for 90 minutes in 1.5-ml microfuge tubes (E&K Scientific, Santa Clara, CA). Reactions were terminated by adding 1 ml of iced binding buffer followed by immediate centrifugation at 20,800g for 10 minutes at 4°C. The supernatant was aspirated, and the pellet was washed twice with 1 ml of iced binding buffer. After aspirating the supernatant of the final wash, the pellet was dissolved in 100 μl of 0.5 M NaOH and then counted in a scintillation counter (LS6500; Beckman Coulter, Fullerton, CA). Results were evaluated using a one-site model (GraphPad Prism 7.0). IC₅₀ values were converted to K_i values as previously described (Cheng and Prusoff, 1973).

In Vitro Reversibility Assay.

Dissociation rates for compounds were determined using conditions similar to those used for equilibrium binding. Briefly, membrane homogenates were combined with an 80% inhibitory concentration of compound (determined by equilibrium binding above) and preincubated for 60 minutes at 37°C with gentle agitation. The reaction was then diluted 15-fold with radioligand to a final concentration of 8 nM. The reaction was terminated at various time points by centrifugation and washed with 10 mM Tris, 1 mM EDTA, and 1% BSA (pH = 7.5), and radioactivity in the pellet was determined as described above. Dissociation rates were determined using a one-phase exponential decay model (GraphPad Prism 7.0).

In Vitro Competitive Activity-Based Protein Profiling Studies.

Human, rat, and mouse tissues were processed to isolate membrane and soluble fractions as described previously (Nomanbhoy et al., 2003). Briefly, brain or liver samples (Analytical Biologic Services) were Dounce-homogenized in 50 mM HEPES (pH = 7.4) containing 320 mM sucrose followed by centrifugation at 1400g for 3 minutes to remove the cellular debris. The supernatant was subjected to ultracentrifugation at 145,000g for 45 minutes to produce the soluble fraction in the supernatant and the pelleted membrane fraction. The membrane fraction was washed in lysis buffer by Dounce-homogenization, reisolated by ultracentrifugation, and resuspended in lysis buffer by Dounce-homogenization. Total protein concentration was assessed for the soluble and membrane fractions using the bicinchoninic acid assay (ThermoFisher Scientific, Waltham, MA). Samples were diluted to 1 mg/ml in lysis buffer, snap frozen on liquid nitrogen, and stored at −80°C until use.

Competitive activity-based protein profiling (ABPP) was used as previously described to examine inhibitor selectivity against the serine hydrolase family (Jessani et al., 2002). Briefly, soluble or membrane proteomes (1 mg/ml) were incubated with inhibitors (0, 1, or 10 μM) for 30 minutes at room temperature under rotation in a final volume of 50 μl lysis buffer containing 10 mM MgCl₂ and DMSO at a final concentration of 2%. FP-Rh probe was then added to the sample at a final concentration of 10 μM and further incubated for 30 minutes at room temperature while rotating. Reactions were quenched by adding 25 μl of 3× SDS-PAGE loading buffer and heating to 75°C for 10 minutes. Proteins were resolved on a 4%–12% Bolt Bis-Tris mini-gel (10 μl sample/lane) at 150 V for 2 hours using 4°C chilled 1× 4-morpholinepropanesulfonic acid buffer and Bolt antioxidant in the front compartment of the gel box (ThermoFisher Scientific). The use of chilled running buffer and antioxidant reagent improved the resolution of serine hydrolases. The Chameleon Duo Pre-stained Protein Ladder was used to mark molecular weights on the gels (LI-COR Biosciences, Lincoln, NE). Upon completion, gels were rinsed with deionized water and scanned for fluorescence using a Typhoon 9500 system, excitation with 532-nm laser, and long pass green emission filter (GE Life Sciences, Marlborough, MA).

In Vitro Selectivity Panel.

JNJ-42226314 selectivity was further assessed in an extensive panel of binding assays including adenosine (A₁, A_2A, A₃), adrenergic (α₁, α₂, α₁), angiotensin (AT₁), dopamine (D₁, D₂), bradykinin (B₂), cholecystokinin (CCK_A), galanin (GAL₂), melatonin (ML₁), muscarinic (M₁, M₂, M₃), neurotensin (NT₁), neurokinin (NK₂, NK₃), opiate (μ, κ, δ), serotonin (5-HT_1A, 5-HT_1B, 5-HT_2A, 5-HT₃, 5-HT_5A, 5-HT₆, 5-HT₇), somatostatin, vasopressin (V_1a), dopamine transporter, norepinephrine transporter, and ion channels (sodium, potassium, calcium, and chloride). The assays were performed by Eurofins (Celles L’Evescault, France).

JNJ-42226314 was also tested against an in-house panel of other receptors, enzymes, and ion channels that could potentially mediate analgesic effects, including human cyclooxygenase 1/cyclooxygenase 2, human transient receptor potential cation channel subfamily V member 1 receptor (TRPV1), rat N-Type calcium channel, canine transient receptor potential cation channel subfamily melastatin member 8 (TRPM8), human and rat FAAH, and human CB1 and CB2.

In Vivo Studies.

All animal experiments were performed in accordance with the Guide for Care and Use of Laboratory Animals adopted by the National Institutes of Health [National Research Council, 2011] and approved by the Janssen Research & Development Institutional Animal Care and Use Committee. Animals were housed under controlled conditions with a 12-hour light/dark schedule and a temperature of 22 ± 2°C. Food and water were provided ad libitum. Experiments were performed after a 1-week acclimation period unless stated otherwise.

Ex Vivo MAGL and CB1 Occupancy Studies.

Experiments were performed in 18 male C57Bl/6 mice weighing 20–30 g (The Jackson Laboratory, Bar Harbor, ME) and 27 male Sprague-Dawley rats weighing 300–400 g (Harlan Laboratories, Livermore, CA). Animals received intraperitoneal injections of JNJ-42226314 or vehicle before time and dose dependency were assessed. The animals were euthanized with CO₂ at varying time points after drug administration (n = 3 per time point or dose). Brains were rapidly removed and frozen on powdered dry ice and stored at −80°C until sectioning. Plasma samples were also collected for bioanalysis of JNJ-42226314 and 2-AG by liquid chromatography–tandem MS (LC-MS/MS). Twenty-micrometer–thick tissue sections at the level of the hippocampus were prepared for autoradiography. MAGL radioligand binding was determined at room temperature with 3 nM [³H] SAR-127303 (20.7 Ci/mmol) in 50 mM Tris containing 1% BSA (pH = 7.4). Sections were incubated for 10 minutes to minimize dissociation. Determination of nonspecific binding was made in the presence of 10 μM KML-29 (Chang et al., 2012). Slides were washed 4 × 10 minutes in incubation buffer followed by two dips in deionized water. Sections were allowed to dry before scanning with a β-Imager (Biospacelab, Nesles la Vallee, France) for 2 hours. Quantitative analysis was performed using the M3 Vision (Biospacelab). Ex vivo MAGL labeling was expressed as the percentage of specific labeling in corresponding brain areas of vehicle-treated animals. The percentage of MAGL occupancy was plotted against time or dose using GraphPad Prism.

A similar procedure was used to measure CB1 occupancy on adjacent sections. Sections were incubated with 10 nM [³H] CP-55940 (164.9 Ci/mmol) (Herkenham et al., 1990), and 10 μM 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-(1-piperidyl)pyrazole-3-carboxamide (Gatley et al., 1996) was used to determine nonspecific binding. Sections were incubated for 10 minutes to minimize dissociation.

In Vivo Microdialysis.

A total of 51 male Sprague-Dawley rats (Charles River Laboratories, Hollister, CA) weighing approximately 300–400 g were used.

For hippocampal 2-AG measurements, a 14-mm guide cannula (MAB 2/6/9.14/IC; SciPro, Sanborn, NY) was surgically implanted into the hippocampus [from bregma: anterior-posterior (AP) = −5.6 mm, medial-lateral (ML) = 5.0 mm, dorsal-ventral = −3.0 mm], and animals were allowed a 1-week recovery period prior to experimentation. On the morning of the experiment, 6-mm shaft/4-mm membrane probes (MAB 6.14.4 PES, 15-kDa molecular weight cutoff; SciPro) were slowly inserted into the cannula. The probes were perfused continuously with 10% HP-β-CD w/v in 1× artificial cerebrospinal fluid at a rate of 0.7 µl/min to optimize 2-AG collection, as described previously (Buczynski and Parsons, 2010). Baseline collection began after an acclimatization period of 60–120 minutes. Samples were collected every 30 minutes into a 96-well plate at 1°C. Each well contained 29 μl of acetonitrile to help retard 2-AG isomerization in the dialysate. Test compounds or vehicle were administered intraperitoneally, and collection continued for 150 minutes. Dialysis samples were analyzed for 2-AG by LC (Shimadzu LC-30AD with CBM-20A system controller) with MS/MS detection. A Kinetex (Phenomenex, Torrance, CA) 1.7 µm XB-C18 100 Å, 2.1 × 100 mm ultra-HPLC column was used with a mobile phase containing a mixture of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The mobile phase was held at an A:B ratio of 80:20 for 1.5 minutes. The step gradient was quickly changed for 2 minutes to an A:B ratio of 30:70; slowly changed over the next 5.5 minutes to an A:B ratio of 25:75; and then quickly changed to A:B ratio of 5:95 in 0.5 minutes, held for 2 minutes, and then returned to starting conditions. The total run time was 12.5 minutes, and the flow rate was 0.35 ml/min. MS/MS detection was performed on a SCIEX 6500 + Q-trap (SCIEX, Framingham, MA) in the positive ion mode by multiple reaction monitoring (MH+/daughter was 379.2 → 287.3 m/z). The detection limit for 2-AG was 0.2 pg/10 μl.

For cortical norepinephrine (NE) measurements, a 4-mm guide cannula was surgically implanted into the medial prefrontal cortex (from bregma: AP = 3.2 mm, ML = 1.0 mm, dorsal-ventral = –1.0 mm), and animals were allowed to recover for 1 week prior to experimentation. On the evening before the experiment, 4-mm shaft/4-mm membrane probes (AJ-4-04, 50-kDa molecular weight cutoff; Amuza, San Diego, CA) were perfused continuously with 1× artificial cerebrospinal fluid at a rate of 1.0 µl/min and were slowly inserted into the cannula. On the morning of the experiment, dialysate samples were collected in a 96-well plate at 4°C and a flow rate of 1.1 µl/min with a collection time of 30 minutes per sample. Test compounds or vehicle were intraperitoneally administered, and collection continued for 150 minutes. Collected samples were analyzed for NE by HPLC with electrochemical detection using an Eicompak CA-5ODS column (2.1 mm i.d. × 150 mm; Amuza) with the graphite electrode potential set to +450 mV against an Ag/AgCl reference electrode. The mobile phase consisted of 100 mM sodium phosphate buffer containing 400 mg/l octanesulphonic acid, 50 mg/l EDTA, and 5% (v/v) methanol (pH = 6.0). The detection limit for NE was below 10 fg/10 μl.

In Vivo Electrophysiology.

A total of 20 male Sprague-Dawley rats (Charles River Laboratories) weighing approximately 300–400 g were used.

Animals were anesthetized with isoflurane (5% for induction, 1.5% for maintenance; 0.9 l/min flow rate) and head-fixed in a stereotaxic apparatus. Body temperature was maintained with a homeothermic heating pad, and respiration, heart rate, and pulse oximetry were continuously monitored. A 2.5-mm piece of skull over the hippocampus was removed, and the exposed dura mater was carefully opened to allow for insertion of a concentric bipolar stimulating electrode (FHC, Bowdoin, ME) and tungsten recording microelectrode (World Precision Instruments, Sarasota, FL) into the Schaffer collateral pathway and pyramidal cell layer of CA1, respectively, using the following stereotaxic coordinates from bregma: stimulating electrode: AP = 3.4 mm, ML = 2.75 mm; recording electrode: AP = 4.4 mm, ML = 2.25 mm. Electrodes were initially inserted to a depth of 2 mm below the pial surface before evoked responses were obtained with test stimuli to aid in determining the final electrode placement.

Stimulation intensity was adjusted to evoke a 30%–60% maximal response, and subsequent stimulus pulses were continuously delivered at 0.33 Hz. Signals were amplified and filtered (1 Hz to 10 kHz, DAM80 bio-amplifier; World Precision Instruments), before collection (40-kHz sampling; PowerLab 16/35 data acquisition unit, LabChart Pro software; ADInstruments, Colorado Springs, CO). A 10-minute period of stable baseline recordings was obtained before compound or vehicle administration. Evoked responses were obtained for an additional 90 minutes thereafter. At the end of most recording sessions, the brain was removed and quickly frozen to determine 2-AG and compound concentrations via LC-MS/MS. The evoked field potential slope was measured and normalized to the mean baseline slope for that animal. Results were then averaged across animals for each dosing group according to time relative to compound or vehicle dosing.

Sleep/Wake Recording and Analysis.

Experiments were conducted in 15 male Sprague-Dawley rats weighing 350–450 g (Harlan Laboratories). Animals were surgically implanted with radio-telemetry probes [PhysioTel F40-EET; Data Sciences International (DSI), St. Paul, MN] to record locomotor activity and body temperature, with two epidural electrodes placed in the frontal and parietal cortex to record the EEG activity and in the dorsal nuchal muscles to record the electromyogram activity. Animals were allowed to recover for 2 weeks before being moved to a room housing receiving platforms to record wireless signals sent from the telemetry probes. EEG and electromyogram signals were recorded and digitized at a sampling rate of 250 Hz, with high and low pass filters set at 0.5 and 60 Hz, respectively, on a PC computer running Dataquest ART software (DSI). Polysomnographic wave forms were analyzed in 10-second periods and visually classified as wake, non–rapid eye movement (NREM) or rapid eye movement (REM) sleep using NeuroScore software (DSI). EEG gamma power during wake was determined by power spectral analysis using a Fast-Fourier transform with a frequency range of 30–60 Hz and was expressed as a percentage of the total power of the recorded signal.

The effects of JNJ-42226314 were assessed in two separate groups of animals (3 and 30 mg/kg), with animals in each group also receiving a control vehicle dosing. A washout period of, at minimum, 3 days and, at maximum, 7 days was observed between JNJ-42226314 and vehicle treatments. In each study, JNJ-42226314 or vehicle was administered by intraperitoneal injection at 8:00 AM, 2 hours after the start of the “light phase,” to correspond to the time of day in which the in vivo electrophysiology experiments were conducted. Signals were recorded for 10 hours afterward. Latency to NREM sleep was defined as the time interval between treatment and the first six consecutive NREM epochs, whereas REM sleep latency was defined as the time interval between treatment and the first two consecutive REM epochs. For each animal, total wake, NREM sleep, and REM sleep time and average wake gamma power were determined over successive 2-hour periods after dosing to provide enough time points for appropriate statistical analysis. Locomotor activity and body temperature were assessed in 1-hour periods. Results were averaged across animals and expressed as the mean ± S.E.M. within each time interval.

Rat Complete Freund’s Adjuvant–Induced Radiant Heat Hypersensitivity.

To assess the ability of JNJ-42226314 to reverse complete Freund’s adjuvant (CFA)-induced thermal hyperalgesia, baseline response latencies on a radiant heat paw stimulator were obtained before an intraplantar injection of 100 μl (1 μg/μl) CFA (1:1 CFA:saline) in 30 male Sprague-Dawley rats (250–350 g), as previously described (Parsons et al., 2015). Withdrawal responses that consisted of quick hind paw movements away from the stimulator were recorded. Paw movements associated with locomotion or a shifting of weight were not counted. Stimulus intensities evoking 10- to 15-second baseline withdrawal latencies were used, with a maximum cutoff of 20 seconds. Hypersensitivity was evaluated 24 hours after CFA administration. Only rats that exhibited at least a 25% reduction in response latency from baseline (i.e., hyperalgesia) were included for further analysis. Blood samples and brains were removed from the rats immediately following the last behavioral measure and analyzed for the concentration of JNJ-42226314 as well as 2-AG.

Chronic Constriction Injury–Induced Cold Hypersensitivity Assay.

To assess the ability of JNJ-42226314 to reverse chronic constriction injury (CCI)-induced cold hypersensitivity, four loose ligatures of 4-0 chromic gut were surgically placed around the left sciatic nerve of 36 male Sprague-Dawley rats (175–325 g) under inhalation anesthesia, as described by Bennett and Xie (1988). Fifteen to 35 days following CCI surgery, animals were placed in elevated observation chambers consisting of wire mesh floors. A series of five applications of acetone (0.10 ml/application) was gently sprayed onto the underside of the paw through the mesh floor using a multidose syringe device. Positive responses consisted of a rapid or protracted withdrawal or lifting of the paw. The percentage of positive responses from the five trials represented the animal’s overall response, which was then averaged within each treatment group. Percent inhibition was then calculated as:

Statistics.

Results are given as the mean ± S.E.M. To assess whether the data deviated from normality, we employed the Shapiro and Wilk’s W test for normality. Data across all experimental replicates for each dose and each time point were tested, with a false discovery rate correction to account for the multiple tests. Treatment effects on field excitatory postsynaptic potentials (fEPSPs), 2-AG/norepinephrine microdialysis area under the curve (AUC) measurements, and CCI-induced cold allodynia were analyzed by one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test to determine significance versus vehicle. Paired t tests were used to assess treatment effects on sleep latencies. Time course data from experiments assessing treatment effects on 2-AG microdialysis, sleep/wake duration, locomotor activity, body temperature, gamma power, and CFA/CCI-induced pain hypersensitivity were analyzed via two-way repeated measures ANOVA (treatment × time as factors) and followed by Bonferroni multiple comparisons post hoc test to determine significance versus vehicle at a given time point. Norepinephrine microdialysis time course data were analyzed with a Linear Mixed-Effects model to compare treatments effects over time with “Treatment” and “Time” as fixed effects and “animal” as a random effect. The information derived from the model was used to make pairwise treatment comparisons with false discovery rate adjusted P values. Differences at P < 0.05 were considered statistically significant. ANOVA and t tests were performed with GraphPad Prism, version 7.0. Linear Mixed-Effects analysis was performed with R software, version 3.5.0 (https://www.r-project.org/).