The mGluR2 positive allosteric modulator BINA decreases cocaine self-administration and cue-induced cocaine-seeking and counteracts cocaine-induced enhancement of brain reward function in rats
Xinchun Jin1*, Svetlana Semenova1*, Li Yang2, Robert Ardecky2, Douglas J. Sheffler3, Russell Dahl2, P. Jeffrey Conn3, Nicholas D. P. Cosford2, and Athina Markou1
1Department of Psychiatry, School of Medicine, University of California San Diego, La Jolla, CA 92093; 2Program on Apoptosis and Cell Death Research,The Burnham Institute for Medical Research, La Jolla, CA 92037; 3Department of Pharmacology, Vanderbilt University Medical Center, Nashville, TN 37232
Running title: mGluR2 positive allosteric modulator and cocaine
Athina Markou, Ph.D., Department of Psychiatry, M/C 0603, School of Medicine, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0603 USA; Tel: +1-858-534-1572; Fax: +1-858-534-9917; E-mail address:
*These authors contributed equally to this work.
SUPPLEMENTARY MATERIALS AND METHODS
Cell Culture
Human Embryonic Kidney (HEK-293) cell lines co-expressing rat mGluR2 or ratmGluR3 and G-protein-coupled inwardly rectifying potassium (GIRK) channels (Niswender et al 2008) were grown in Growth Media containing 45% Dulbecco's modified Eagle’s medium (DMEM), 45% Ham's F12, 10% fetal bovine serum (FBS), 20 mM HEPES(pH 7.3), 1 mM sodium pyruvate, 2 mM L-glutamine, 1x antibiotic/antimycotic, 1x non-essential amino acids, 700 μg/ml G418 (Mediatech, Inc., Herndon, VA), and 0.6 μg/ml puromycin dihydrochloride (Sigma-Aldrich, St. Louis, MO) at 37ºC in the presence of 5% CO2. All cell culture reagents were purchased from Invitrogen Corp. (Carlsbad, CA) unless otherwise specified. Rat mGluR2 and mGluR3 cell lines were prepared by polymerase chain reaction (PCR) amplification of the entire coding sequence of each receptor and cloning into pIRES puro 3 (Invitrogen) at the NotI site. HEK/GIRK/M4 cells (Niswender et al 2008) were transfected with 24 μg of either DNA, and stable transfectants were selected with puromycin. Polyclonal cell lines were established for mGluR2/GIRK and mGluR3/GIRK.
Thallium flux assays
Compound activity at the rat Group II mGluRs (mGluR2 and mGluR3) was assessed using thallium flux through GIRK channels as previously described (Niswender et al 2008). Briefly, cells were plated into 384-well, black-walled, clear-bottomed poly-D-lysine-coated plates (Becton Dickinson, Bedford, MA) at a density of 15,000 cells/20 μl/well in DMEM containing 10% dialyzed FBS, 20 mM HEPES, and 100 units/ml penicillin/streptomycin (Assay Media). Plated cells were incubated overnight at 37°C in the presence of 5% CO2. The following day, the medium was removed from the cells, and 20 μl/well of 1.7 μM BTC-AM, an indicator dye (Invitrogen; prepared as a stock in DMSO and mixed in a 1:1 ratio with Pluronic acid F-127) prepared in Assay Buffer (Hanks’ balanced salt solution [Invitrogen] containing 20 mM HEPES, pH 7.3) was added to the plated cells. Cells were incubated for 1 h at room temperature, and the dye was replaced with 20 μl/well of assay buffer. Test compounds prepared in assay buffer were added at 2x final concentration followed 2.5 min later by agonist addition via a kinetic imaging plate reader (FDSS 6000; Hamamatsu Corporation, Bridgewater, NJ). Agonists were diluted in thallium buffer (125 mM sodium bicarbonate, 1 mM magnesium sulfate, 1.8 mM calcium sulfate, 5 mM glucose, 12 mM thallium sulfate, 10 mM HEPES) at 5x the final concentration to be assayed. Data were analyzed as previously described (Niswender et al 2008).
BINA synthesis
General methods
All reactions were carried out under a nitrogen atmosphere, and glassware and syringes were routinely dried in an electric oven at 100ºC. All solvents used were commercially available from Aldrich or Acros. 1H NMR spectra (300 or 400 MHz) were recorded in either DMSO-D6 or CD3OD. Chemical shifts are reported in parts per million downfield from the internal standard Me4Si. Liquid chromatography mass spectrometry (LCMS) analyses were carried out on a Shimadzu LCMS 2010 Series LC System with a Kromasil 100 5m C18 column (502.1 mmID). For flash chromatography, silica gel 60 (230-400 mesh, Merck) was employed.
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2-Cyclopentyl-1-(4-methoxy-2,3-dimethylphenyl)ethanone (3)
To a suspension of 2,3-dimethyl anisole 1 (9.82 g, 0.0716 mol) and aluminum chloride (14.23 g, 0.107 mol) in dichloromethane (200 ml) at room temperature was slowly added a solution of 2-cyclopentylacetyl chloride 2 (9.18 g, 0.107 mol) in dichloromethane (50 ml) over 1.5 h. The reaction was stirred overnight at room temperature. The reaction mixture was poured into a 2 L beaker containing ice (500 g) and water (500 ml). The organic layer was separated, washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel with dichloromethane to givecompound 3 (17.40 g, 98% yield) as a colorless oil. 1H NMR (300 MHz, DMSO-D6) δ 1.05-1.18 (bm, 2H), 1.44-1.66 (bm, 4H), 1.70-1.78 (bm, 1H), 2.07 (s, 3H), 2.04-2.11 (bm,2H), 2.18 (s, 3H), 2.23-2.33 (bm, 2H), 3.76 (s, 3H), 6.83 (t,1H), 7.02 (t, 1H) (see Fig. S1 for synthetic scheme).
2-Cyclopentyl-1-(4-methopxy-2,3-dimethylphenyl)prop-2-en-1-one (4)
In a 200 ml round bottom flask was placed compound 3 (9.60 g, 0.039 mol), dimethylamine hydrochloride (14.31 g, 0.176 mol) and glacial acetic acid (1.5 ml). The slurry was heated at 100ºC for 1 h.Dry dimethylformamide (45 ml) was added, and the mixture was heated overnight at 100ºC. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (200 ml) and water (200 ml). The organic layers were separated and washed successively with water (200 ml), saturated sodium bicarbonate, 1 M hydrochloric acid, and saturated sodium chloride solutions. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel with dichloromethane:hexane (1:1) to give compound 4 (4.07 g, 40% yield) as a white solid. 1H NMR (300 MHz, DMSO-D6) δ 1.05-1.18 (bm, 2H), 1.44-1.66 (bm, 4H), 1.70-1.78 (bm, 1H), 2.09 (s, 3H), 2.04-2.11 (bm, 1H), 2.17 (s, 3H), 2.23-2.33 (bm, 1H), 3.76 (s, 3H), 5.44 (d, J = 6.2 Hz, 1H), 5.85 (d, J = 6.2 Hz, 1H), 6.88 (m,1H), 7.11 (m, 1H).
2-Cyclopentyl-5-methoxy-6,7-dimethyl-2,3-dihydro-1H-inden-1-one (5)
Compound 4 (8.90 g, 0.035 mol) was slowly added to concentrated sulfuric acid (35 ml) at room temperature. The reaction was stirred for 4 h at room temperature and then poured into a 1 L beaker containing ice (500 g). The water/ice layer was extracted with ethyl acetate (200 ml), and the organic layer was washed successively with water (200 ml), saturated sodium bicarbonate, and saturated sodium chloride solutions. The residue was purified by column chromatography over silica gel with dichloromethane to give compound 4 (6.67 g, 74% yield) as a white solid. 1H NMR (400 MHz, CD3OD) δ 1.05-1.18 (bm, 2H), 1.44-1.76 (bm, 4H), 1.90-1.98 (bm, 2H), 2.11 (s, 3H), 2.24-2.31 (bm, 3H), 2.54 (s, 3H), 3.05-3.18 (bm, 1H), 3.90 (s, 3H), 6.82 (s, 1H).
2-Cyclopentyl-5-hydroxy-6,7-dimethyl-2,3-dihydro-1H-inden-1-one (6)
Compound 5 (6.02 g, 0.0248 mol) was added to a mixture of glacial acetic acid (100 ml) and 48% HBr (100 ml) at room temperature. The solution was heated at reflux for 4 h and then slowly cooled to room temperature. Compound 6 crystallized out of the reaction mixture and was collected and washed with ice cold diethyl ether and air dried to yield compound 6 (5.14 g, 85 %) as an off white solid. 1H NMR (300 MHz, DMSO-D6) δ 1.05-1.20 (bm, 2H), 1.44-1.81 (bm, 4H), 1.85-1.96 (bm, 2H), 2.11 (s, 3H), 2.24-2.35 (bm, 3H), 2.34 (s, 3H), 3.05-3.18 (bm, 1H), 6.82 (s, 1H).
Methyl 3’-methylbiphenyl-4-carboxylate (9)
To 4-(methoxycarbonyl)phenylboronic acid 7 (17.98 g, 0.01 mol) and 3-iodotoluene 8 (21.80 g, 0.01 mol) dissolved in toluene (400 ml), ethanol (400 ml), and 2M sodium carbonate solution (200 ml) was added tetrakis(triphenylphosphine)palladium(0) (0.5 g). The reaction mixture was purged with nitrogen and then heated at reflux overnight. The reaction mixture was cooled to room temperature, and ethyl acetate (400 ml) was added.The organic layers were separated and washed successively with water (200 ml), saturated sodium bicarbonate solution, 1 M hydrochloric acid, and saturated sodium chloride solution. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel with dichloromethane:hexane (1:1) to give compound 9 (18.20 g, 80.4% yield) as an off-white solid. 1H NMR (300 MHz, DMSO-D6) δ 2.39 (s, 3H), 3.87 (s, 3H), 7.21 (m, 1H), 7.39 (m, 1H), 7.45-7.65 (m, 2H), 7.82 (d, J = 8.0 Hz, 2H), 8.03 (d, J = 8.0 Hz, 2H).
Methyl 3’-(bromomethyl)biphenyl-4-carboxylate (10)
To a suspension of compound 9 (22.70 g, 0.01 mol) and N-Bromosuccinimide (NBS, 17.79 g, 0.01 mol) in carbon tetrachloride (600 ml) was added 2,2'-Azobis(isobutyronitrile)(AIBN, 0.2 g). The mixture was heated at reflux overnight during which time the reaction mixture became clear. The reaction was cooled to room temperature and filtered. The filtrate was washed with carbon tetrachloride (50 ml), andthe organic layer was concentrated to dryness under vacuum to give compound 10 (30.1 g, 98% yield) as an off-white solid. This compound was used without further purification. 1H NMR (300 MHz, DMSO-D6) δ 3.92 (s, 3H), 4.83 (s, 2H), 7.46-7.55 (m, 1H), 7.71-7.83 (m, 1H), 7.75-7.85 (m, 2H), 7.89 (d, J = 8.0 Hz, 2H), 8.11 (d, J = 8.0 Hz, 2H).
Methyl 3’-([2-cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5yloxy]methyl)biphenyl-4-carboxylate (11)
To a solution of compound 10 (4.0 g, 0.0131 mol) and compound 6 (3.28 g, 0.0131 mol) in dry dimethylformamide (50 ml) was added potassium carbonate (27.6 g, 0.02 mol). This suspension was heated to 85ºC for 8 h. The reaction was cooled and filtered, and the filtrate was washed with ethyl acetate (100 ml). The combined organic layers were evaporated under reduced pressure. The residue was dissolved in ethyl acetate (400 ml), and the organic layer was washed successively with water (200 ml), saturated sodium bicarbonate solution, 1 M hydrochloric acid, and saturated sodium chloride solution. The organic layer was dried over sodium sulfate and concentrated under reduced pressure. The residue was purified by column chromatography over silica gel with dichloromethane:ethyl acetate (100:0 to 90:10 gradient) to give compound 11 (4.10 g, 67% yield) as a white solid. 1H NMR (300 MHz, DMSO-D6) δ 0.95-1.05 (bm, 2H), 1.44-1.65 (bm, 4H), 1.70-1.91 (bm, 1H), 2.14 (s, 3H), 2.04-2.11 (bm, 1H), 2.18 (s, 3H), 2.23-2.33 (bm, 2H), 2.61-2.82 (m, 1H), 3.01-3.15 (m, 1H), 3.89 (s, 3H), 5.31 (s, 2H), 7.08 (s, 1H), 7.55-7.58 (m,3H), 7.79-7.88 (m, 2H), 8.01-8.08 (m, 2H).
3’-([2-Cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5yloxy]methyl)biphenyl-4carboxylic acid (12)
To a solution of compound 11 (4.10 g, 0.0088 mol) in ethanol (50 ml) was added 2 M LiOH (10 ml). This solution was heated at reflux overnight under nitrogen. The reaction was cooled, and the pH of the solution was adjusted to pH 1 with 1 M HCl. To this mixture was added ethyl acetate (200 ml), and the organic layer was separated, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure to yield compound 12 (3.95 g, 98% yield) as a white solid. 1H NMR (300 MHz, DMSO-D6) δ 0.95-1.05 (bm, 2H), 1.44-1.65 (bm, 4H), 1.70-1.91 (bm, 1H), 2.14 (s, 3H), 2.04-2.11 (bm, 1H), 2.18 (s, 3H), 2.23-2.33 (bm, 2H), 2.61-2.82 (m, 1H), 3.01-3.15 (m, 1H), 3.89 (s, 3H), 5.31 (s, 2H), 7.07 (s, 1H), 7.52-7.59 (m,2H), 7.70-7.75 (m, 1H), 7.79-7.91 (m, 3H), 8.01-8.09 (m, 2H).
Potassium 3’-([2-cyclopentyl-6-7-dimethyl-1-oxo-2,3-dihydro-1H-inden-5yloxy]methyl)biphenyl-4-carboxylate (13)
To a solution of compound 12 (3.95 g, 0.0087 mol) dissolved in methanol (100 ml) was added 1 M KOH (8.7 ml, 0.0087 mol), and the solution was stirred for 1 h at room temperature. To this solution was added water (200 ml), and the entire mixture was frozen with liquid nitrogen and lyophilized overnight to give compound 13 (4.20 g, 98% yield) as a yellow solid. 1H NMR (400 MHz, DMSO-D6) δ 0.95-1.05 (bm, 2H), 1.44-1.65 (bm, 4H), 1.70-1.91 (bm, 1H), 2.14 (s, 3H), 2.04-2.11 (bm, 1H), 2.18 (s, 3H), 2.23-2.33 (bm, 2H), 2.61-2.82 (m, 1H), 3.01-3.15 (m, 1H), 3.89 (s, 3H), 5.19 (s, 2H), 7.07 (s, 1H), 7.42-7.52 (m,2H), 7.52-7.57 (d, J = 8.0 Hz, 2H), 7.71-7.77 (m,1H), 7.78 (s, 1H), 7.79-7.91 (d, J=8.0 Hz, 2H), MS m/e (ES+ mode), 455.14 found, 455.24 calculated. See Fig. S2-S4 for 1H NMR and HPLC/MS spectra.
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Absorption, distribution, metabolism, and excretion (ADME) and pharmacokinetic studies
In vitro assays
Microsomal stability assay. Pooled rat liver microsomes (BD Biosciences, #452701) were preincubated with test compounds at 37.5°C for 5 min in the absence of nicotinamide adenine dinucleotide phosphate (NADPH). The reaction was initiated by the addition of NADPH and then incubated under the same conditions. The final incubation concentrations were 4 M test compound, 2 mM NADPH, and 1 mg/ml (total protein) liver microsomes in phosphate-buffered saline (PBS), pH 7.4. One aliquot (100 l) of the incubation mixture was withdrawn at 15 min time-points and combined immediately with 200 l of ACN/MeOH containing an internal standard. After mixing, the sample was centrifuged at approximately 13000 rpm for 12 min. The supernatant was transferred to an autosampler vial, and the amount of test compound was quantified using a Shimadzu LCMS 2010EV mass spectrometer (Shimadzu Scientific Instruments, Columbia, MD). The change of the area-under-the-curve (AUC) of the parent compound as a function of time was used as a measure of microsomal stability. Test compounds were tested in duplicate with verapamil (microsomal t1/2 = 11 min) as the positive control.
Plasma stability assay. A 20 µl aliquot of a 10 mM solution of the test compound in dimethyl sulfoxide (DMSO) was added to 2.0 ml of heparinized rat plasma (Lampire, P1-150N) to obtain a 100 µM final solution. The mixture was incubated for 1 h at 37.5°C. Aliquots of 100 µl were taken (0 min, 30 min, 1 h) and diluted with 200 µl of MeOH containing internal standard. After mixing, the sample was centrifuged at approximately 13000 rpm for 12 min. The supernatant was transferred to an autosampler vial, and the amount of test compound was quantified using a Shimadzu LCMS-2010EV system. The change of the AUC of the parent compound as a function of time was used as a measure of plasma stability.
Parallel artificial membrane permeation assay—blood-brain barrier. A 96-well microtiter plate (Millipore, #MSSACCEPTOR) was completely filled with aqueous buffer solution (pH 7.4) and covered with a microtiter filterplate (Millipore, #MAPBMN310). The hydrophobic filter material was impregnated with a 10% solution of polar brain lipid extract in chloroform (Avanti, #141101C) as the artificial membrane, and the organic solvent was allowed to completely evaporate. Permeation studies were started by the transfer of 200 l of a 100 M test compound solution on top of the filterplate. Generally, phosphate buffer (pH 7.2) was used. The maximum DMSO content of the stock solutions was < 1.5%. In parallel, an equilibrium solution lacking a membrane was prepared using the exact concentrations and specifications but lacking the membrane. The concentrations of the acceptor and equilibrium solutions were determined using the Shimadzu LCMS-2010EV and AUC methods. The acceptor plate and equilibrium plate concentrations were used to calculate the permeability rate (LogPe) of the compounds. Assays were run in triplicate, returning three effective permeability coefficients (Pe) for each compound. The LogPe values were calculated using the following equation:
LogPe = log (C • -ln[1 – (drug)Acceptor / (drug)equilibrium])
where C = (VD • VA) / ([VD + VA] area • time)
Parallel artificial membrane permeation assay—gastrointestinal. A 96-well microtiter plate was completely filled with aqueous buffer solution (pH 7.4) and covered with a microtiter filterplate (polyvinylidene fluoride). The hydrophobic filter material was impregnated with 5 l of an artificial membrane solution consisting of phosphatidylcholine in n-dodecane (5% w/v, Avanti Polar Lipids). Permeation studies were started by the transfer of 200 l of a 100 M test compound solution (PBS, < 2% DMSO) to the donor plate. In parallel, an equilibrium solution lacking a membrane was prepared using the exact concentrations and specifications but lacking the membrane. The plates were incubated, unshaken, at room temperature. After 20 h, the two plates were separated, and the concentrations of BINA in the acceptor and equilibrium plates were determined using the Shimadzu LCMS-2010EV in selective ion monitoring mode and AUC methods. The acceptor and equilibrium plate concentrations were used to calculate the permeability rate (LogPe) of the compounds. Assays were run in triplicate along with two reference standards (warfarin, experimental LogPe = -3.71; verapamil, experimental LogPe = -3.05). The LogPevalues were calculated using the following equation:
LogPe = log (C • -ln[1- (drug)Acceptor / (drug)equilibrium])
where C = (VD • VA) / ([VD + VA] area • time)
In vivo pharmacokinetic analysis
Plasma sample preparation. Naive rats (n = 4 per dose) were treated with BINA at doses of 20 or 40 mg/kg (i.p.). Blood samples (400µl) from the tail vein were collected at severaltime-points (1, 2, 3, 4, 6, and 24 h). Plasmawas separated from the red cells by centrifugation and frozen at –20°C until assayed. BINA was extracted from the plasma samples using a two-fold excess of acetonitrile. The extracts were vortexed for 3 min and centrifuged for 15 min at 7500 g, after which the supernatants were collected to be assayed by liquid chromatography-mass spectrometry (LCMS).
In a subsequent experiment, naive rats (n = 5 per dose) were treated with BINA (20 and 40 mg/kg). Rats’ brains, as well as plasma samples, were collected 1 h after BINA administration to determine BINA concentration in the brain and blood at the optimal time-point of BINA plasma concentration determined by the previous experiments.
Brain sample preparation. Whole rat brains were homogenized for 6 min. The homogenized samples were extracted with a water:acetonitrile (25:75) solution containing 1% formic acid. The samples were vortexed for 3 min and centrifuged for 15 min at 7500 g, after which the supernatants were collected. Prior to analysis, the samples were diluted in a 50% (v/v) solution of acetonitrile in water and filtered and analyzed by LCMS.
Analysis and determination of pharmacokinetic parameters. Concentrations of BINA in plasma and brain extracts were determined using a validated analytical procedure based on high-performance liquid chromatography (HPLC). LCMS analyses were carried out using a Shimadzu LCMS-2010EV operating in electrospray ionization mode. Chromatography was carried out using gradient elution (water-acetonitrile) on a Kromisil C18 reverse-phase column at a flow rate of 1 ml/min. Plasma compound concentrations were determined using a seven-point calibration curve derived from peak areas obtained from serially diluted solutions of BINA using Shimadzu LabSolutions software and Microsoft Excel. Pharmacokinetic parameters were determined by noncompartmental analysis using PK Solutions version 2.0 software (Summit Research Services).
Intracranial self-stimulation apparatus, surgery, and procedure
Training and testing occurred in 16 sound-attenuated plexiglas chambers (San Diego Instruments, San Diego, CA) that contained a metal wheel manipulandum. Brain stimulation was delivered by constant current stimulators (San Diego Instruments, San Diego, CA). Subjects were prepared with bipolar stainless steel electrodes (Plastics One, Roanoke, VA) in the posterior lateral hypothalamus (anterior/posterior, -0.5 mm from bregma; lateral, ±1.7 mm; dorsal/ventral, -8.3 mm from dura) (Pellegrino et al., 1986) with the incisor bar elevated 5.0 mm above the interaural line.
The ICSS procedure was a modification of a procedure originally developed by Kornetsky and colleagues (Kornetsky et al., 1979). This discrete-trial current-threshold procedure has been described in detail by Markou and Koob (Markou and Koob, 1991; 1992). At the start of each trial, rats received a noncontingent electrical stimulus (100 Hz rectangular cathodal pulses). During the following 7.5 s limited hold (if the rats responded by turning the wheel manipulandum; positive response) they received a second contingent stimulus identical to the previous noncontingent stimulus. During a 2 s period immediately after a positive response, further responses had no consequences. If no response occurred during the 7.5 s limited hold, a negative response was recorded. The intertrial interval (ITI), which followed the limited hold period, had an average duration of 10 s (7.5-12.5 s). Responses occurring during the ITI resulted in a further 12.5 s delay of the onset of the next trial. Stimulation intensities varied according to the psychophysical method of limits. Thus, rats received four alternating series of ascending and descending current intensities starting with a descending series. Within each series, the stimulus intensity was altered by 5 A steps between each set of trials (3 trials/set). The initial stimulus intensity was set at 30-40 A above the baseline current threshold for each rat. A test session typically lasted 30 min and provided brain reward thresholds as the dependent variable. The threshold for each descending series was defined as the stimulus intensity between a successful completion of a set of trials (two consecutive sets), during which the rat failed to respond positively on two or more of the three trials for two consecutive steps. For the ascending series, the reverse situation defined the threshold. Thus, current thresholds were recorded for each of the four series, and the mean of these values was taken as the threshold for that session. Baseline reward thresholds were considered stable when less than 10% variation occurred over 5 consecutive days. The latency between the onset of the noncontingent stimulus at the start of each trial and a positive response was recorded as the response latency. The response latency for each test session was defined as the mean response latency of all trials during which a positive response occurred.