Animals.

Adult male Sprague-Dawley rats (body weight of 300–400 g during the conduct of experiments; Harlan, Indianapolis, IN) were individually housed for behavioral studies and housed under standard conditions for neurochemical and pharmacokinetic assays. Upon arrival, rats were given free access to food and water in their home cages, which were maintained at 24°C, 45% humidity, and 14/10-hour light/dark cycle. Rats acclimated to the environment for 1 week prior to initiation of experiments, and when used in behavioral experiments, rats were handled daily. During operant training, food in the home cage was limited to 5–10 g/day to maintain bodyweight at approx. 85%, and then free feeding continued once rats reached criteria for stable responding. Experiments were conducted during the light phase. Experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Kentucky and were in accordance with the 2011 National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Chemicals.

[³H]Dopamine ([³H]DA; dihydroxyphenylethylamine, 3,4-[7-³H]; specific activity, 24.8 Ci/mmol), [³H]5-hydroxytryptamine ([³H]5-HT; 5-hydroxytryptamine creatinine sulfate, 5-[1,2-³H[N]]; specific activity, 29.5 Ci/mmol), [³H]nicotine ([³H]NIC; (L-(-)-[N-methyl-³H]; specific activity, 80.4 Ci/mmol), and Microscint 20 cocktail were obtained from PerkinElmer Inc. (Waltham, MA). [³H]Dofetilide ([N-methyl-³H]; specific activity, 80 Ci/mmol) and [³H]methyllycaconitine ([³H]MLA; [1α,4S,6β,14α,16β]-20-ethyl-1,6,14,16-tetramethoxy-4-[[[2-([3-³H]-[3-³H]-methyl-2,5-dioxo-1-pyrrolidinyl)benzoyl]oxy]methyl]-aconitane-7,8-diol; specific activity, 60 Ci/mmol) were obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). (+)-Methamphetamine hydrochloride, 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine dihydrochloride (GBR-12935), amitriptyline, cytisine, fluoxetine, S(-)-nicotine hydrogen tartrate salt, nomifensine, 1-octanesulfonic acid sodium salt, 3-(4-methoxyphenyl)propanoic acid, 5-hydroxytryptamine creatinine sulfate, adenosine 5′-triphosphate magnesium salt (ATP-Mg²⁺), α-d-glucose, ammonium chloride, anhydrous sodium sulfate, ascorbate oxidase, catechol, celite, dichloromethane, diethyl ether, dimethylformamide, dopamine hydrochloride, ethyl acetate, EDTA, ethylene glycol tetraacetate (EGTA), hexane, hydrochloric acid, hydroxybenzotriazole, lithium aluminum hydride (LiAlH₄), Kolliphor EL, magnesium sulfate, methanesulfonyl chloride, methanol, methylene chloride, HEPES, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride, pargyline hydrochloride, phenyllithium, polyethyleneimine (PEI), potassium hydroxide, potassium tartrate dibasic hemihydrate, R-propylene oxide, sodium azide, silica, sodium chloride, sodium hydroxide, sucrose, tetrahydrofuran, triethylamine, triphenylphosphine, tris[hydroxymethyl]aminomethane base, tetrahydrofuran, and tris[hydroxymethyl]-aminomethane hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO). Acetonitrile, calcium chloride, citric acid, formic acid, hydrogen chloride, methanol, monopotassium phosphate, potassium chloride, sodium bicarbonate, and sodium hydroxide were obtained from Fisher Scientific Co. (Pittsburgh, PA). Ascorbic acid, 1% non-essential amino acids, and scintillation cocktail 3A70B were purchased from AnalaR-BHD Ltd. (Polle, UK), Thermo Fisher Scientific (Waltham, MA), and Research Products International Corp. (Mount Prospect, IL), respectively. Minimum essential medium, Hanks’ Balanced Salt solution, and 10% fetal bovine serum were obtained from Gibco (Grand Island, NY). (2R,3S,11bS)-2-Ethyl-3-isobutyl-9,10-dimethoxy-2,2,4,6,7,11b-hexahydro-1H-pyrido[2,1-a]isoquinolin-2-ol (RO4-1284) was a generous gift from Hoffmann-LaRoche Inc. (Nutley, NJ).

GZ-11608 Synthesis.

To a solution of phenyllithium (50 ml, 1.8 M in dibutyl ether) in tetrahydrofuran (50 ml) was added R-propylene oxide (5 g) dropwise at −78°C. The resulting mixture was stirred at −78°C for 1 hour before being warmed to room temperature and stirred overnight. The reaction was quenched by addition of saturated aqueous ammonium chloride solution (50 ml). The aqueous phase was extracted with ethyl acetate (3 × 50 ml) and the combined organic layers were washed with brine (100 ml), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was chromatographed on silica (hexanes/ethyl acetate 10:1 to 3:1) to afford R-1-phenylpropan-2-ol (10.4 g) as a colorless oil. To a solution of R-1-phenylpropan-2-ol (4.16 g, 30.5 mmol) and triethylamine (7.72 g, 10.64 ml, 76.27 mmol) in dichloromethane (100 ml) was added methane sulfonyl chloride (4.54 g, 3.07 ml, 39.65 mmol) dropwise at 0°C. The resulting mixture was stirred at 0°C for 15 minutes. Dichloromethane (100 ml) was added to the mixture, which was then washed with water (2 × 150 ml). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The resulting light-yellow oil was mixed with sodium azide (5.95 g, 91.5 mmol) in dimethylformamide (40 ml) and the mixture heated at 55°C for 3 hours. The reaction mixture was diluted with diethyl ether (150 ml) and washed with water (2 × 100 ml) and brine (100 ml). The organic layer was separated, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was chromatographed on silica (hexanes/ethyl acetate 50:1 to 20:1) to afford S-(2-azidopropyl)benzene (4.38 g) as a colorless oil. To a solution of S-(2-azidopropyl)benzene (4.0 g, 24.81 mmol) in tetrahydrofuran (90 ml) and water (10 ml) was added triphenylphosphine (9.11 g, 34.74 mmol) at room temperature. The resulting mixture was stirred for 18 hours and water (50 ml) was added. Hydrochloric acid (1.0 M) was then added to the mixture to obtain a pH 1.0, and the aqueous phase was extracted with diethyl ether (3 × 100 ml) and dichloromethane (2 × 100 ml). Aqueous sodium hydroxide (15% w/v) was added dropwise to the aqueous phase to adjust the pH to 11 and the solution extracted with dichloromethane (5 × 60 ml). The combined, separated organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to afford S-1-phenylpropan-2-amine as a colorless oil. The crude amino product (3.03 g, 22.41 mmol) was mixed with 3-(4-methoxyphenyl)-propanoic acid (4.44 g, 24.65 mmol), and hydroxybenzotriazole (3.63 g, 26.89 mmol) in dichloromethane (60 ml) at room temperature. Triethylamine (5.67 g, 56.03 mmol) was added followed by N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (5.15 g, 26.89 mmol). The resulting mixture was stirred overnight. The reaction mixture was diluted with dichloromethane (100 ml) and washed with water (3 × 50 ml) and brine (50 ml). The organic layer was separated and dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was chromatographed on silica (dichloromethane/ethyl acetate 10:1) to afford S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propanamide (6.18 g) as a white solid. To a solution of S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propanamide (420 g, 1.41 mmol) in tetrahydrofuran (7 ml), LiAlH₄ (5.6 ml, 1.0 M in tetrahydrofuran) was added dropwise at 0°C. The resulting mixture was heated at reflux for 3 hours before being cooled to 0°C. The reaction was quenched by careful addition of water (0.21 ml), followed by 15% w/v aqueous sodium hydroxide (0.21 ml) and water (0.63 ml). The resulting milky suspension was warmed to room temperature, anhydrous magnesium sulfate was added, and the mixture was stirred for 2 hours, filtered through a pad of Celite, and rinsed with ethyl acetate. The combined filtrates were concentrated under vacuum, and the crude product was chromatographed on silica (methylene chloride/methanol, 30:1 to 10:1) to afford GZ-11608 (380 mg, 95%) as a white solid with low melting point: ¹H NMR (400 MHz, CDCl₃) δ 7.15–7.34 (m, 5H), 7.01 (d, J = 8.4 Hz, 2H), 6.80 (dd, J = 8.4, 2.0 Hz, 2H), 3.77 (s, 3H), 2.87 (m, 1H), 2.47–2.77 (m, 6H), 1.73 (m, 2H), 1.05 ppm (d, J = 6.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ 157.9, 139.7, 134.2, 129.5, 129.4, 128.6, 126.4, 113.9, 55.4, 54.8, 46.8, 43.8, 32.8, 32.1, 20.4 ppm; MS (EI) m/z 282.2 [M-1]⁺; purity: >97% liquid chromatography–mass spectrometry (LC-MS). GZ-11608 was converted to hydrochloride salt by treating with HCl in ether (2 M). The salt form was used for all the experiments.

Vesicular [³H]DA Uptake.

To obtain the affinity (K_i value) of GZ-11608 for VMAT2, the ability of GZ-11608 to inhibit [³H]DA uptake into isolated synaptic vesicles in a concentration-dependent manner was determined, as previously described (Teng et al., 1997). Nonspecific uptake of [³H]DA was determined in the presence of RO4-1284 (10 μM). Briefly, striata from individual rats were homogenized in 14 ml of ice-cold 0.32 M sucrose solution containing 5 mM sodium bicarbonate (pH 7.4) with 10 up-and-down strokes of a Teflon pestle homogenizer (clearance approx. 0.009 inch) using a Maxima Digital Overhead Stirrer (400 rpm; Fisher Scientific Co.). Homogenates were centrifuged (2000g for 10 minutes at 4°C), and the resulting supernatants were centrifuged (10,000g for 30 minutes at 4°C). Pellets were resuspended in 2 ml of 0.32 M sucrose solution containing 5 mM sodium bicarbonate (pH 7.4) and were subjected to osmotic shock by transfer of samples to tubes containing 7 ml of ice-cold MilliQ water. Samples were homogenized on ice with five up-and-down strokes of a Teflon pestle homogenizer. After 5 minutes, osmolarity was restored by transferring the samples to tubes containing 900 μl of 0.25 M HEPES and 900 μl of 1.0 M potassium tartrate dibasic hemihydrate solution. Samples were centrifuged (20,000g for 20 minutes at 4°C) and resulting supernatants centrifuged (55,000g for 1 hour at 4°C). To the resulting supernatants, 100 μl of 10 mM magnesium sulfate, 100 μl of 0.25 M HEPES, and 100 μl of 1.0 M potassium tartrate dibasic hemihydrate solution were added, followed by a final centrifugation (100,000g for 45 minutes at 4°C). Final pellets were resuspended in 2.4 ml of assay buffer (25 mM HEPES, 100 mM potassium tartrate dibasic hemihydrate, 50 μM EGTA, 100 μM EDTA, 1.7 mM ascorbic acid, and 2 mM ATP-Mg²⁺, pH 7.4 adjusted dropwise with 10 M potassium hydroxide). Aliquots of the resulting suspension of isolated synaptic vesicles (100 μl) were added to tubes containing assay buffer (300 μl), one of a range of concentrations of GZ-11608 (final concentration 0.1 nM to 0.1 mM; 50 μl) and 0.1 μM [³H]DA (final concentration 10 nM; 50 μl) to obtain a final assay volume of 500 μl. After incubation for 8 minutes in a 37°C water bath (Reciprocal Shaking Bath Model 50; Precision Scientific, Chicago IL), [³H]DA uptake was stopped by rapid filtration through presoaked (0.5% PEI for 1 hour at 4°C) Whatman GF/B Glass microfiber filters (1.0 μm pore size; Clifton, NJ) via a cell harvester (MP-43RS; Brandel Inc., Gaithersburg, MD). Subsequently, filters were washed three times with 4 ml of ice-cold wash buffer (25 mM HEPES, 100 mM potassium tartrate dibasic hemihydrate, 50 μM EGTA, 100 μM EDTA, 1.7 mM ascorbic acid, and 2 mM magnesium sulfate; pH 7.4 adjusted dropwise with 10 M potassium hydroxide). Scintillation cocktail (5 ml) was added to tubes containing the filters, followed by shaking for 30 minutes at room temperature. Radioactivity retained on the filters was determined by liquid scintillation spectrometry (TRI-CARB 2100 TR Packard scintillation counter; Packard BioScience Company, Meriden, CT).

Synaptosomal [³H]DA and [³H]5-HT Uptake.

To evaluate the selectivity of GZ-11608 at VMAT2 relative to DAT and serotonin transporter (SERT), GZ-11608 inhibition of [³H]DA and [³H]5-HT uptake, respectively, into rat striatal synaptosomes was determined using previously published methods (Teng et al., 1997; Norrholm et al., 2007). Briefly, striata from individual rats were homogenized in 20 ml of 0.32 M sucrose containing 5 mM sodium bicarbonate (pH 7.4) with 16 up-and-down strokes of a Teflon pestle homogenizer (clearance approx. 0.003 inch) using the Maxima Digital Overhead Stirrer (400 rpm). Homogenates were centrifuged (2000g for 10 minutes at 4°C). Supernatants were centrifuged (20,000g for 17 minutes at 4°C) and pellets were resuspended (2.4 ml for DAT assay; 1.4 ml for SERT assay) in Krebs’ buffer (125 mM sodium chloride, 5 mM potassium chloride, 1.5 mM magnesium sulfate, 1.25 mM calcium chloride, 1.5 mM monopotassium phosphate, 10 mM α-D-glucose, 25 mM HEPES, 0.1 mM EDTA, 0.1 mM pargyline hydrochloride, and 0.1 mM ascorbic acid, and saturated with 95% O₂/5% CO₂; pH 7.4 adjusted dropwise with 1 M sodium hydroxide). For DAT and SERT assays, aliquots of synaptosomal suspension (25 and 50 μl, respectively) were added to tubes containing Krebs’ buffer (375 and 125 μl, respectively) and one of a range of concentrations of GZ-11608 (0, 0.1 nM to 0.1 mM) in 50 and 25 μl buffer, respectively. Uptake in the absence of GZ-11608 represents control. For nonspecific uptake, assay tubes contained nomifensine (final concentration, 100 μM in 50 μl for DAT assays) and fluoxetine (final concentration, 10 μM in 25 μl for SERT assays) in the absence of GZ-11608. For SERT assays, GBR-12935 (final concentration, 100 nM in 25 μl), a DAT inhibitor, was added to all assay tubes to prevent [³H]5-HT uptake into dopaminergic terminals (Norrholm et al., 2007). DAT and SERT assay tubes (450 and 225 μl, respectively) were incubated at 34°C for 5 minutes. After incubation, tubes were placed on ice for 2 minutes. [³H]DA (final concentration, 10 nM in 50 μl) or [³H]5-HT (final concentration, 10 nM in 25 μl) was added to each tube. DAT and SERT assay tubes (final assay vol, 500 and 250 μl, respectively) were incubated at 34°C for 10 minutes. Uptake was stopped by addition of 3 ml of ice-cold assay buffer and subsequent filtration. [³H]DA or [³H]5-HT retained on the filters (presoaked in assay buffer containing 1 mM catechol for 1 hour at 4°C) was determined as previously described.

[³H]Dofetilide Binding.

GZ-11608 inhibition of [³H]dofetilide binding to hERG assessed potential cardiotoxicity and selectivity of GZ-11608 for VMAT2 over hERG. HEK-293 cells stably expressing hERG channel protein were purchased from Millipore (Catalog number CYL3006; Billerica, MA). Binding assays were performed as previously described (Sviripa et al., 2014; Nickell et al., 2017). Briefly, frozen cells were thawed at 37°C and placed in T-75 cm² flasks (Becton Dickinson and Company, Franklin Lakes, NJ), containing 20 ml of complete media (minimum essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, and 400 μg/ml geneticin), according to the Millipore protocol. For 4–8 hours, cells adhered to flasks in a humidified atmosphere (5% CO₂ at 37°C), after which the media was replaced with 20 ml fresh media. Subsequently, media was replaced every 48 hours. For routine passages, media was removed, cells rinsed with 2 ml of phosphate-buffered saline (137 mM sodium chloride, 2.7 mM potassium chloride, 10 mM disodium hydrogen phosphate, 2 mM potassium dihydrogen phosphate), followed by addition of Hank’s balanced salt solution containing trypsin (0.5 g/l, porcine trypsin) and EDTA (0.5 mM). To dissociate the cells, flasks were placed in a 37°C incubator for 2–5 minutes, and then fresh complete media (5 ml) was added to the cell resuspensions, followed by seeding onto new flasks at 2–3 × 10⁶ cells/flask. Passages were conducted every 6 days. At least three passages occurred before cell membrane collection. On the last passage prior to membrane preparation, cells were seeded onto 150- × 25-mm culture dishes at 2.5 × 10⁶ cells/dish, and culture dishes were incubated (5% CO₂ at 37°C) for 40–48 hours. Media was removed and then culture dishes rinsed twice with 30°C Hanks’ balanced salt solution (13 ml). A solution of ice-cold 0.32 M sucrose with 5 mM sodium bicarbonate (20 ml, pH 7.4) was then added to each culture dish on ice. Cells were scraped gently from the dishes and then homogenized (30 seconds) on ice with a Teflon pestle (approx. 0.003 inch) using a Maximal Digital homogenizer (280 rpm). Homogenates were centrifuged (300 and 800g for 4 minutes each at 4°C). Pellets were resuspended in 9 ml ice-cold MilliQ water, and osmolarity restored by addition of 1 ml of 500 mM Tris buffer (pH 7.4). Samples were centrifuged (20,000g for 30 minutes at 4°C). Pellets were resuspended in 2 ml assay buffer (50 mM Tris, 10 mM potassium chloride, and 1 mM magnesium chloride, pH 7.4, at 4°C). Aliquots of membrane suspension were stored at –80°C until use. To perform the [³H]dofetilide binding assay, membrane suspension was thawed and protein content determined using a Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA), with bovine albumin (Sigma-Aldrich Corporation) as the standard. Duplicate tubes were prepared containing membrane suspension (5 μg/100 μl), one of a range of concentrations of GZ-11608 (final concentrations 0, 0.1 nM to 0.1 mM in 25 μl) or amitriptyline (0, 0.1 nM–0.1 mM; positive control), assay buffer (150 μl), and [³H]dofetilide (5 nM in 25 μl) for a final assay volume of 250 μl. Amitriptyline (1 mM) was used to determine nonspecific binding (Teschemacher et al., 1999; Jo et al., 2000). Samples were incubated for 1 hour at room temperature. Reactions were stopped by rapid filtration through Whatman GF/B Glass microfiber filters presoaked in 0.5% PEI for 1 hour at 4°C. Filters were washed three times with 1 ml ice-cold assay buffer. Radioactivity retained by the filters was determined as previously described.

[³H]NIC and [³H]MLA Binding.

To evaluate the selectivity of GZ-11608 for VMAT2 over α4β2 and α7 nicotinic acetylcholine receptors (nAChRs), GZ-11608 inhibition of [³H]NIC and [³H]MLA binding was determined, respectively, using previously published methods ((Horton et al., 2011a)Horton et al., 2011a). In brief, whole brains excluding cortex and cerebellum from individual rats were homogenized for 90 seconds in 20 vol of ice-cold assay buffer (2 mM HEPES, 14.4 mM sodium chloride, 0.15 mM potassium chloride, 0.2 mM calcium chloride, and 0.1 mM magnesium sulfate, pH 7.5 adjusted dropwise with 1 M sodium hydroxide) using a polytron. Homogenates were centrifuged (31,000g for 17 minutes at 4°C). Pellets were resuspended in 20 vol of assay buffer by sonication (Vibra Cell; Sonics & Materials Inc., Danbury, CT). Subsequently, duplicate samples were incubated in a 37°C water bath for 10 minutes, and then centrifuged (31,000g at 4°C for 17 minutes). Resulting pellets were resuspended in 20 vol of assay buffer by sonication, and centrifuged (31,000g for 17 minutes at 4°C). Final pellets were resuspended and stored in 10 ml of incubation buffer (20 mM HEPES, 144 mM sodium chloride, 1.5 mM potassium chloride, 2 mM calcium chloride, and 1 mM magnesium sulfate, pH 7.5 adjusted dropwise with 1 M sodium hydroxide) at −20°C until use. Thawed membrane suspensions (100–140 μg protein/100 μl) were added to tubes containing one of seven to nine concentrations of GZ-11608 (final concentration, 0, 0.1 nM to 0.1 mM in 50 μl) or nicotine (final concentration, 10 pM to 100 μM; positive control) or methyllycaconitine (final concentration, 10 pM to 100 μM; positive control), and [³H]NIC or [³H]MLA (final concentration, 3 nM in 50 μl), and incubation buffer (50 μl) for a final assay volume of 250 μl. Nonspecific binding of [³H]NIC and [³H]MLA was determined in the presence of 10 μM of cytisine (50 μl) and 10 μM of nicotine (50 μl), respectively. Samples were incubated for 1 hour at room temperature. Unifilter-96 GF/B filter plates (1.0 μm pore size; PerkinElmer, Inc.) were presoaked in 0.5% PEI for 1 hour at 4°C. Reactions were stopped by filtration using a Packard Filter Mate Harvester (Perkin Elmer, Inc.). Plates were washed three times with 350 μl of ice-cold assay buffer, and dried for 1 hour at 45°C. Plates were bottom-sealed, and each well filled with 40 μl Microscint 20 cocktail. Bound radioactivity on the filter was determined via liquid scintillation spectrometry (Top Count NXT scintillation counter; PerkinElmer, Inc.).

Methamphetamine-Evoked [³H]DA Release.

To further evaluate GZ-11608 efficacy as an inhibitor of the pharmacological effects of methamphetamine, the concentration-dependent effect of GZ-11608 to inhibit methamphetamine-evoked dopamine release from isolated striatal synaptic vesicles was determined using previously described methods (Teng et al., 1997; Horton et al., 2013). Also, the underlying mechanism of GZ-11608 inhibition was determined. Initially, the effect of GZ-11608 to release DA from synaptic vesicles was determined. Vesicles were prepared as described above for the vesicular DA uptake assay, except that final pellets were resuspended in a smaller volume (2.7 ml) of assay buffer. To preload the vesicles, [³H]DA (final concentration, 0.3 μM in 300 μl) was added to the vesicle suspension, and incubation proceeded at 37°C for 8 minutes. Samples were placed on ice for 2 minutes to stop [³H]DA uptake, and then centrifuged at 100,000g for 1 hour at 4°C to remove free [³H]DA not transported into the vesicles. Pellets were resuspended in a final volume of 4.2 ml of assay buffer. Aliquots of [³H]DA-preloaded vesicular suspension (180 μl) were added to tubes containing 1 of 11 concentrations of GZ-11608 (final concentrations, 0, 0.1 nM to 0.1 mM in 20 μl). Samples were incubated at 37°C for 8 minutes. Reactions were stopped by addition of 2.5 ml of ice-cold buffer (25 mM HEPES, 100 mM potassium tartrate, 50 μM EGTA, 100 μM EDTA, 1.7 mM ascorbic acid, 2 mM magnesium sulfate, pH 7.4), followed by rapid filtration onto PEI-presoaked GF/B filters, and rinsing of the filters with ice-cold buffer (three times, 4 ml each). Scintillation cocktail was added, and radioactivity retained on the filters determined by liquid scintillation spectrometry. GZ-11608-evoked vesicular DA release was determined as the amount of [³H]DA retained by the vesicles subtracted from the amount retained in control vesicles not exposed to GZ-11608. To determine methamphetamine-evoked [³H]DA release and GZ-11608-induced inhibition of methamphetamine-evoked [³H]DA release, [³H]DA preloaded vesicles (180 μl) were incubated for 8 minutes at 37°C with 1 of 11 methamphetamine concentrations (final concentrations, 0, 0.1 μM to 20 mM in 10 μl) in the absence (control) and presence of a single concentration of GZ-11608 (final concentrations, 0, 10, 500 nM, and 10 μM in 10 μl) in a total volume of 200 μl. Reactions were stopped, radioactivity retained on the filter determined, and vesicular [³H]DA release calculated as described above to determine GZ-11608-induced inhibition of methamphetamine-evoked [³H]DA release.

Methamphetamine Sensitization.

As a rapid means of determining if GZ-11608 decreases the in vivo effects of methamphetamine, locomotor sensitization following repeated methamphetamine injection was the initial assay employed (Alvers et al., 2012; Lee et al., 2018). Repeated methamphetamine administration results in a robust and stable increase in locomotor activity from day-to-day, allowing for reliable evaluation of the ability of compound to reduce the effects of methamphetamine on behavior. Locomotor activity was measured in a locomotor chamber (24 × 24 × 30 cm) with clear acrylic walls and floor. A horizontal 16 × 16 grid of photo beams was located 7 cm above the chamber floor, with each beam 2.5 cm apart. Movement in the chamber resulted in beam breaks, which were recorded and transformed into distance traveled (centimeter) by Versamax and Digipro System software (AccuScan Instruments Inc., Columbus, OH). The effect of GZ-11608 on methamphetamine-sensitized activity was determined using a mixed factor design with methamphetamine as a between-subjects factor and GZ-11608 as within-subjects factor. Rats were assigned randomly to methamphetamine or saline groups. After a week of acclimation, rats were habituated to the apparatus by being placed in the chamber for 1 hour with no injection (day 0). On days 1–10, rats were injected (subcutaneous, s.c.) daily with either methamphetamine (1 mg/kg) or saline (1 ml/kg), immediately placed in the chamber, and activity measured for 1 hour. Methamphetamine dose and number of daily injections were chosen to provide stable, sensitized locomotor activity, on the basis of previous findings (Lee et al., 2018). On day 11, GZ-11608 (0, 1–30 mg/kg, s.c.) in a quasi-randomized dose order was injected 15 minutes prior to the daily methamphetamine or saline injection, and then, rats were placed immediately into the chamber for 1 hour. A washout period (2 to 3 days) intervened between testing of GZ-11608 doses to avoid potential drug accumulation. On washout days, methamphetamine or saline was injected and locomotor activity determined.

In a separate experiment employing a mixed factor design, the effect of GZ-11608 administration by oral gavage (p.o.) on methamphetamine locomotor sensitization was determined. Following repeated methamphetamine or saline for 5 days, rats were habituated to the oral gavage procedure. On days 5–10, rats received vehicle [15% (v/v) Kolliphor EL in saline, p.o., 3 ml] 15 minutes prior to methamphetamine or saline injection (subcutaneous) and were placed in the activity chamber for 1 hour (Wilmouth et al., 2013). On days 11–27, GZ-11608 (0, 17–300 mg/kg, p.o., ascending dose order) was administered, followed 15 minutes later by either methamphetamine or saline injection (1 ml/kg, s.c.), depending on group assignment, and immediate placement into the chamber for 1 hour. Between GZ-11608 doses, 2 to 3 days of washout occurred. On washout days, vehicle was administered by oral gavage and methamphetamine or saline was injected subcutaneously.

Striatal Dopamine Content.

To determine whether GZ-11608 alters striatal dopamine content and/or exacerbates methamphetamine-induced striatal dopamine depletion, GZ-11608 was administered subcutaneously to rats in the absence and presence of a methamphetamine dose known to deplete rat striatal dopamine content (Bowyer et al., 1992, 1994; pilot study). The dose of GZ-11608 was chosen on the basis of its ability to reliably decrease methamphetamine-sensitized locomotor activity. Following acclimation to the colony and 3 days prior to drug injection, a thermal transponder (Bio Medic Data Systems, Inc., Seaford, DE) was implanted (subcutaneously) beneath the scapula to monitor body temperature. In the first series of experiments using a between-groups design, GZ-11608 (17 mg/kg, s.c.) or saline (1 ml/kg, s.c.) was administered 15 minutes prior to methamphetamine (30 mg/kg, i.p.) or saline (1 ml/kg, i.p.), according to random assignment to treatment group. In the second series of experiments also using a between-groups design, GZ-11608 or saline was administered 15 minutes after methamphetamine or saline. Methamphetamine was administered at 22°C ambient temperature in both series of experiments. Body temperature was monitored every 30 minutes for 8 hours following methamphetamine injection. If the body temperature increased to 41.3°C or higher, rats were transferred to a cage placed on ice until body temperature decreased to 40.0°C or below (Bowyer et al., 1992, 1994; Fukumura et al., 1998). Striata were obtained 72 hours after methamphetamine injection and were processed via high-performance liquid chromatography with electrochemical detection. Striata were weighed, placed in 1 ml of perchloric acid and sonicated. Tissue samples were centrifuged at 31,000g for 30 minutes at 4°C. Supernatant (50 μl) was injected onto the octadecyl silica Ultrasphere C18 reverse-phase column (80 × 4.6 mm, 3 μm; ESA Inc., Chelmsford, MA) via autosampler (508; Beckman Coulter, Inc., Fullerton, CA). Dopamine was detected by a coulometric-II detector with guard cell (model 5020; ESA, Inc.) maintained at +0.60 V and an analytical cell (model 5011) maintained at E1 = +0.05 V and E2 = +0.35 V. Mobile phase (MP) consisted of 0.07 M citrate, 0.1 M acetate buffer with 175 mg/l octylsulfonic acid-sodium salt, 650 mg/l of sodium chloride, and 7% methanol (pH 4.2). Flow rate was 1.2 ml/min, and 4–5 minutes were required to analyze each sample. Dopamine standards were used to identify and quantify dopamine peak and amount using 32 Karat software (Beckman Coulter, Inc.).

Methamphetamine Self-Administration.

The ability of GZ-11608 to dose dependently decrease the reinforcing effect of methamphetamine was determined using a within-subject design. Owing to the high doses of GZ-11608 required in the methamphetamine sensitization studies and the assumed low oral bioavailability, subcutaneous rather than oral administration was employed for self-administration studies. Two-lever operant chambers were used to train rats once daily for 3 days in 1-hour sessions to press a lever (active lever) for food pellet reinforcement (45 mg pellet, #F0021; BIO-SERV, Frenchtown, NJ) on a fixed ratio 1 (FR1) schedule, whereas responding on the other lever (inactive lever) had no programmed consequence. Subsequently, the operant schedule was incremented to an FR3 schedule for 3 days, then an FR5 schedule for 14 days until rats met the criteria for stable responding, which included: 1) ≥10 pellets earned/session and 2) a minimum of a 2:1 ratio of active/inactive lever presses. After delivery of each food reinforcer, the lights above both levers were illuminated for a 20-second signaled timeout period. After reaching stable responding for food on the FR5 schedule, rats underwent catheter implantation surgery. Rats were anesthetized (75 mg/kg ketamine, 7.5 mg/kg xylazine, and 0.75 mg/kg acepromazine; i.p.) and a silastic catheter was implanted into the jugular vein. The free end of the catheter was affixed with dental acrylic to the skull by metal screws and exited through the scalp. Rats were allowed to recover for 1 week. Prior to the start and at the end of each behavioral session. Catheters were flushed daily with 0.1 ml heparinized saline to maintain patency. Following recovery from surgery, rats were trained to press a lever for intravenous methamphetamine (0.05 mg/kg per infusion) during 1-hour daily sessions using a standard two-lever procedure as previously reported (Harrod et al., 2001; Beckmann et al., 2012). The FR schedule was incremented across training sessions (3 days, FR1; 3 days, FR3; 14 days, FR5). A 20-second signaled timeout occurred after each methamphetamine infusion. Upon reaching criteria for stable responding (≥10 infusions/session and a 2:1 ratio of active/inactive lever presses), a dose of GZ-11608 (0, 1, 3, 10, 17, and 30 mg/kg, in ascending dose order, s.c.) was administered 15 minutes prior to the session. GZ-11608 vehicle (0 mg/kg) was 15% Kolliphore in saline (1 ml/kg, s.c.).

Food-Maintained Responding.

To evaluate the specificity of the GZ-11608-induced decrease in responding for intravenous methamphetamine, the ability of GZ-11608 to decrease food-maintained responding was determined using a within-subject design. GZ-11608 doses and pretreatment time were as described for methamphetamine self-administration experiments. Experiments were conducted as described above, with the exceptions that no surgery was performed and rats did not self-administer methamphetamine. Instead, rats were trained to a terminal FR5 to respond for food pellets (45 mg pellet, #F0021; BIO-SERV) in daily 1-hour sessions.

Repeated GZ-11608 Administration.

A within-subject design was used to determine if tolerance developed to the GZ-11608-induced decrease in methamphetamine self-administration and/or food-maintained responding. One group of rats underwent operant training for food reinforcement, catheter implantation surgery, and operant training for intravenous methamphetamine (0.05 mg/kg per infusion) self-administration as described above. In these experiments, GZ-11608 (30 mg/kg, s.c.; a dose that reliably decreased methamphetamine self-administration) was administered 15 minutes prior to seven consecutive, daily methamphetamine self-administration sessions. Then, for five consecutive daily sessions, responding for methamphetamine was determined without GZ-11608 treatment. Another group of rats was trained for food-maintained responding, and the effect of repeated GZ-11608 (30 mg/kg, s.c.) was determined using the same dose and pretreatment time as described above for methamphetamine self-administration.

Surmountability.

To determine whether increasing the unit dose of methamphetamine would surmount the effect of GZ-11608 to decrease intravenous methamphetamine self-administration, another group of rats underwent operant training for food reinforcement, catheter implantation surgery, and operant training for methamphetamine (0.05 mg/kg per infusion) self-administration as described above. A within-subject design was employed to establish the methamphetamine dose-response across a range of methamphetamine doses (0.01–0.25 mg/kg per infusion) in the absence of GZ-11608. Each dose of methamphetamine was tested for three consecutive sessions. Then, the methamphetamine dose-response was re-evaluated in the same group of rats following treatment with GZ-11608 (30 mg/kg, s.c.) 15 minutes prior to the session. To maintain stable responding, two intervening maintenance sessions occurred between each session in which GZ-11608 was administered. For these maintenance sessions, no GZ-11608 treatment was administered prior to self-administration of each methamphetamine unit dose.

Reinstatement.

The ability of GZ-11608 to decrease cue- and methamphetamine-induced reinstatement of methamphetamine-seeking behavior was determined using previously published methods (Harrod et al., 2003). In brief, three groups of rats were trained to self-administer methamphetamine (0.05 mg/kg per infusion) as described above, except that cue lights were illuminated for 5 seconds at the beginning of each session prior to presentation of the levers. For cue-induced reinstatement experiments, upon reaching the criteria for stable responding, rats underwent extinction for 14 days. During extinction, cue lights were not illuminated at the beginning or during daily 1-hour sessions, and active lever presses did not result in methamphetamine infusion. The day after the last extinction day (test for reinstatement), the cue light was illuminated at the beginning and during the session, and the dose effect for GZ-11608 to decrease cue-induced drug-seeking behavior was determined. Because drug-seeking behavior is diminished with repeated testing, two groups of rats were needed to generate the complete dose response. In one group, low doses (0, 3, 5.6, and 10 mg/kg, s.c.) were evaluated in a randomized order 15 minutes prior to the session, and higher doses (0, 10, and 17 mg/kg, s.c.) were evaluated in a second group. To maintain responding at extinction levels, five intervening sessions occurred between each session in which GZ-11608 was administered. On intervening sessions, there was no cue light illumination, no GZ-11608 pretreatment, and no methamphetamine infusion.

The effect of GZ-11608 (0, 10, 17, and 30 mg/kg, s.c.; in randomized order) on methamphetamine-induced reinstatement of drug-seeking behavior was determined in a third group of rats. GZ-11608 or saline was injected 15 minutes prior to the session, and methamphetamine (0.5 mg/kg, i.p.) was injected immediately prior to the session to reinstate drug-seeking behavior. Experiments were conducted using procedures similar to those in the experiments evaluating cue-induced reinstatement of drug seeking, except that the 20-second contingent cue light illumination continued during the 14 days of extinction, as well as on reinstatement tests.

Substitution of GZ-11608 for Methamphetamine.

To determine whether GZ-11608 substitutes for methamphetamine in rats trained to self-administer intravenous methamphetamine, another experiment was conducted using a mixed factor design with GZ-11608 treatment as a between-subject factor, and dose and session as within-subject factors, similar to previously published methods (Harrod et al., 2003; Beckmann et al., 2012). Rats were trained to stable performance for methamphetamine (0.05 mg/kg per infusion) self-administration under an FR5 schedule of reinforcement as described above. Upon reaching the criteria for stable responding, rats were assigned randomly to either the GZ-11608 or saline groups. For the GZ-11608 group, responding on the active lever under the FR5 schedule resulted in intravenous infusions of GZ-11608 (0.01, 0.05, 0.1, and 0.5 mg/kg per infusion; in ascending order), each dose was administered across four consecutive sessions. Only saline was available (intravenous) to the saline group across the same number of self-administration sessions. Then, for both GZ-11608 and saline groups, methamphetamine (0.05 mg/kg per infusion) was available for four consecutive self-administration sessions.

GZ-11608 Self-Administration.

To determine whether GZ-11608 was self-administered in drug naive rats, a mixed factor design with GZ-11608 treatment as a between-subject factor, and dose and session as within-subject factors was conducted, similar to previously published methods (Harrod et al., 2003). Rats underwent operant training for food reinforcement, catheter implantation surgery, and random assignment to operant training of GZ-11608 or saline intravenous self-administration. For the saline group, only intravenous saline was available across the experiment. For the GZ-11608 group, each GZ-11608 dose (0.5, 0.1, and 0.05 mg/kg per infusion) was available in a descending order on an FR1 schedule of reinforcement for five consecutive days, followed by availability on an FR2 schedule for 3 days. To maintain stable responding, intervening maintenance sessions occurred after the evaluation of each GZ-11608 dose. During the maintenance sessions, rats responded for food reinforcement under FR1 for 2 days, and then under FR2 for 1 day; GZ-11608 was not available. As a positive control, ability to self-administer methamphetamine (0.05 mg/kg per infusion) under an FR1 for 5 days, FR2 for 3 days, and FR5 for 10 days was determined in the GZ-11608 group. To further evaluate GZ-11608 as a reinforcer, responding for intravenous GZ-11608 (0.1 mg/kg per infusion) or saline (depending on random group assignment) was determined in drug-naive rats under an FR1 for 5 days, FR2 for 3 days, and FR5 schedule for 10 days.

Pharmacokinetics.

For dosing and pharmacokinetic sample collection, GZ-11608 was formulated in 15:85 Kolliphor EL/saline and was administered by oral gavage, intravenous bolus injection, or subcutaneous injection. Blood samples were collected in heparinized tubes from the saphenous vein and centrifuged at 4300g for 2 minutes. Plasma was collected and immediately frozen on dry ice followed by storage at −80°C until processing and analysis. An internal standard (ISTD) solution of 250 ng/ml of 1,4-diphenethylpiperidine (GZ-361B) in 50:50 methanol/Milli-Q water (v/v) was prepared from a stock solution of 100 μg/ml in water. Independent stocks of GZ-11608 (100 μg/ml in water) were prepared and used for calibrators and quality control samples. Ten spiking solutions [in 50:50 methanol/water (v/v)] were used for calibration curves ranging from 2.5 to 1000 ng/ml. Likewise, spiking solutions were used to prepare quality control samples at 7.5, 25, 500, and 850 ng/ml. Calibration and quality control samples consisted of 10 μl of spiking solution and 50 μl blank rat plasma. Experimental plasma samples were thawed on ice (2–4°C), briefly vortexed, and diluted by the addition of 10 μl of sample and 10 μl of 50:50 methanol/water into 40 μl of blank rat plasma. Internal standard (15 μl of the 250 ng/ml ISTD solution) was added to samples and vortex mixed. All calibrators, quality control, and experimental samples were processed by protein precipitation with the addition of 2.4× volume of methanol (180 μl) containing 0.1% formic acid, vortex mixed, stored at –20°C for 20 minutes, and centrifuged at 21,000g for 20 minutes at 4°C. Resulting supernatants were transferred into high-performance liquid chromatography vials for analyses.

For LC-MS analyses, samples were injected (10-μl volume) via an autosampler (15°C) and were analyzed for the positive-mode m/z 284.2/121.1 and 294.2/105.2 transitions of GZ-11608 and ISTD, respectively, on an AB Sciex Triple TOF (qTOF) 5600 unit (AB Sciex, Framingham, MA) equipped with a Shimadzu Prominence HPLC system (Shimadzu, Columbia, MD) controlled by Analyst TF software (ver. 1.7). Analyte and ISTD were resolved chromatographically (6 and 6.37 minutes, respectively). An Inertsil ODS-3 (4 μm, 3 × 100 mm) analytical column (GL Sciences, Rolling Hills Estates, CA) with a guard column of the same stationary phase was used. Analytes eluted using a gradient of aqueous 0.1% formic acid (MP-A) and acetonitrile (MP-B; gradient conditions: 30% MP-B to 75% MP-B linear ramp over 3 minutes, then held at 75% MP-B for 1 minute, followed by 3 minutes linear ramp back to 30% MP-B and equilibration at 30% MP-B for 5 minutes) at a 0.2 ml/min total flow rate. Eluent was routed through the qTOF TurboIonSpray ESI probe thru an integrated Valco diverter valve (VICI Valco Instruments, Huston, TX) between 5 and 7.3 minutes and vaporized/ionized using a source temperature of 550°C and an ion spray voltage of 4500 V (gas settings at GS1 = 35/GS2 = 35/CUR = 30). qTOF method consisted of two repeat experiments scanning the 100–300 amu mass range for product ions of GZ-11608 (m/z 284.15; DP = 80/CE = 35/CES = 0/IRD = 67/IRW = 25) or ISTD (m/z 294.2; DP = 70/CE = 35/CES = 0/IRD = 67/IRW = 25). Quantitation was conducted using MultiQuant software (ver. 3.0; AB Sciex) on the extracted 121.1 and 105.2 m/z product ion peak areas, with calibration curves constructed from the analyte to ISTD peak area and concentration ratios (best fit line from linear regression weighted at 1/Y²). Concentrations of GZ-11608 in experimental samples were interpolated from the corresponding curve; otherwise, samples outside the calibration range were reprocessed at 1:4 or 1:10 dilution with blank rat plasma alongside likewise diluted and processed quality control. Samples were analyzed following injection of a calibration curve and quality control samples. Three of four quality control samples were required to be within 15% of nominal values. Quality controls were injected every 24 experimental samples and at the end of the sample sequence. The lower limit of quantitation was 2.5 ng/ml and the upper limit of quantitation was 1000 ng/ml. Accuracy and precision were within 15%.

Brain Tissue Collection and Processing for HPLC with Tandem Mass Spectrometry Analysis.

Following administration of GZ-11608 (30 mg/kg, s.c.), rats were placed under isoflurane (USP; Henry Schein, Dublin, OH) anesthesia and prepared for perfusion with 1:99 (v/v) heparin in 0.9% saline as previously described (Gage et al., 2012). Brains were collected at 5, 30, and 45 minutes, and at 1, 3, 6, 8, and 12 hours after dosing. To remove systemic blood, rats were perfused at 20 ml/min until the liver was cleared of blood. The brain was removed with the aid of rongeurs and homogenized 1:2 (w/v) with ice-cold phosphate-buffered saline (137 mM sodium chloride, 2.7 mM potassium chloride, 1.0 mM sodium hydrogen phosphate, and 1.8 mM potassium dihydrogen phosphate). Homogenates were stored at −80°C until analysis. Calibration, quality control, and experimental brain samples were thawed and vortex mixed. A 50-μl aliquot was added to 15 μl of internal standard (250 ng/ml GZ-361B in methanol/water 50:50). Samples were treated with 200 μl of 0.1% formic acid in methanol to precipitate proteins. Samples were vortex mixed (10 seconds), chilled (−20°C for 20 minutes), remixed (10 seconds), and centrifuged (13,000g for 18 minutes at 4°C). Resulting supernatants were collected into amber HPLC vials fitted with 150-μl glass inserts, and immediately analyzed for analyte content by LC with tandem mass spectrometry as described for the plasma analyses.

Data Analysis.

Specific [³H]DA uptake, [³H]5-HT uptake, [³H]dofetilide binding, [³H]NIC binding, and [³H]MLA binding were calculated by subtracting nonspecific uptake or binding from total uptake or binding, respectively. The GZ-11608 concentration that produced 50% inhibition of specific uptake or binding (IC₅₀ values) was obtained from individual concentration-response curves via an iterative curve-fitting program (Prism 7.03; GraphPad Software, Inc., La Jolla, CA). Inhibition constants (K_i values) were determined using the Cheng-Prusoff equation (Cheng and Prusoff, 1973). The selectivity ratio for GZ-11608 relative to the off-target sites was determined as the K_i value for inhibition of VMAT2 divided by the K_i value for DAT, SERT, hERG, and nAChRs, respectively. EC₅₀ values from individual concentration-response curves for methamphetamine or GZ-11608 to evoke [³H]DA release from synaptic vesicles was determined using Prism 7.0. The mechanism of GZ-11608-induced inhibition of methamphetamine-evoked vesicular [³H]DA release was determined using Schild analysis. Dose ratios (DR) were obtained by dividing the EC₅₀ for methamphetamine-evoked [³H]DA release in the presence of each concentration of GZ-11608 by that in the absence of GZ-11608. Log (DR-1), plotted as a function of log GZ-11608 concentration, provided the Schild regression; linearity of the slope was significantly different from unity if the 95% confidence intervals (CI) did not include 1.0 (Prism 7.03; Kenakin et al., 2006).

For all behavioral experiments, distance traveled and number of responses were subjected to repeated measures analysis of variance (ANOVA), followed by Tukey’s post-hoc tests when appropriate, unless otherwise indicated.

Pharmacokinetic data were evaluated graphically using GraphPad (Prism 8.0) and analyzed using Phoenix 64 (Certara USA, Inc., Princeton, NJ) to estimate pharmacokinetic parameters. Noncompartmental methods were used to estimate C_max, time of maximum concentration (T_max), area under the curve (AUC_0→∞), elimination half-life (t_1/2), and clearance or clearance/F, where F is the bioavailability. Bioavailability estimates of the oral and subcutaneous routes were determined using the respective AUCs and doses in comparison with the intravenous bolus data using the following equation:(1)Where i.v. and e.v. denote intravenous and extravascular routes. Linear regression was performed to estimate whether the slope of dose-versus-PK parameters deviated from zero (GraphPad).