Discriminative Stimulus Properties of the Endocannabinoid

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Discriminative Stimulus Properties of the Endocannabinoid Catabolic Enzyme Inhibitor SA-57 in Mice
Robert A. Owens, Bogna Ignatowska-Jankowska, Mohammed Mustafa, Patrick M. Beardsley, Jenny L. Wiley, Abdulmajeed Jali, Dana E. Selley, Micah J. Niphakis, Benjamin F. Cravatt, Aron H. Lichtman
Affiliations: R.A.O.; B.I.J.; M.M.; P.M.B.; A.J.; D.E.S.; A.H.L. Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA
J.L.W: RTI International, 3040 Cornwallis Road, Research Triangle Park, NC 27709-2194, USA
M.J.N.; B.F.C.: The Skaggs Institute for Chemical Biology and Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA 92037, USA

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a) Running Title: Endocannabinoid Discriminative Stimulus

b) Corresponding author: Aron H. Lichtman, PO Box 980613, Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia 23298-0613, USA; [email protected]

c) Number of text pages: 15 Number of figures: 10 Number of tables: 2 Number of references: 65 Abstract number of words: 236 Introduction number of words: 747 Discussion number of words: 1,359

d) List of nonstandard abbreviations

2-arachidonoylglycerol (2-AG); cannabinoid-1 (CB1) receptor; cannabinoid-2 (CB2) receptor; CP55,940

((-)-cis-3-[2-Hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol); fatty

acid amide hydrolase (FAAH); monoacylglycerol lipase (MAGL); alpha/beta-hydrolase domain 6

(ABHD6); N-arachidonoyl ethanolamine (anandamide; AEA); rimonabant (5-(4-chlorophenyl)-1-(2,4-

dichlorophenyl)-4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide); PF-3845 (N-3-pyridinyl-4-[[3-

[[5-(trifluoromethyl)-2-pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) JZL184 (4-[Bis(1,3-

benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester); JZL195 (4-

nitrophenyl 4-(3-phenoxybenzyl)piperazine-1-carboxylate); MJN110 (2,5-dioxopyrrolidin-1-yl 4-(bis(4-

chlorophenyl)methyl)piperazine-1-carboxylate);

SA-57

(4-[2-(4-Chlorophenyl)ethyl]-1-

piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester); SR144,528 (N-[(1S)-endo-1,3,3-

trimethylbicyclo

[2.2.1]heptan2-yl]-5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-1H-

pyrazole-3-carboxamide); URB597 (Cyclohexylcarbamic acid 3'-(Aminocarbonyl)-[1,1'-biphenyl]-3-yl

ester); KT195 ([4-(4′-Methoxy[1,1′-biphenyl]-4-yl)-1H-1,2,3-triazol-1-yl](2-phenyl-1-piperidinyl)-

methanone); KT182 ([4-[3′-(Hydroxymethyl)[1,1′-biphenyl]-4-yl]-1H-1,2,3-triazol-1-yl](2-phenyl-1-

piperidinyl)-methanone).

e) Recommended section: Behavioral pharmacology

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Abstract Whereas inhibition of fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL), the respective major hydrolytic enzymes of N-arachidonoyl ethanolamine (AEA) and 2arachidonoylglycerol (2-AG) elicits no or partial substitution for Δ9-tetrahydrocannabinol (THC) in drug discrimination procedures, combined inhibition of both enzymes fully substitutes for THC, as well as produces a constellation of cannabimimetic effects. The present study tested whether C57BL/6J mice would learn to discriminate the dual FAAH-MAGL inhibitor SA-57 (4-[2-(4-chlorophenyl)ethyl]-1piperidinecarboxylic acid 2-(methylamino)-2-oxoethyl ester) from vehicle in the drug discrimination paradigm. In initial experiments, 10 mg/kg SA-57 fully substituted for CP55,940 ((-)-cis-3-[2-hydroxy4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol), a high efficacy CB1 receptor agonist in C57BL/6J mice and for AEA in FAAH (-/-) mice. The majority (i.e., 23 of 24) of subjects achieved criteria for discriminating SA-57 (10 mg/kg) from vehicle within 40 sessions, with full generalization occurring 1-2 h post injection. CP55,940, the dual FAAH-MAGL inhibitor JZL195 (4nitrophenyl 4-(3-phenoxybenzyl)piperazine-1-carboxylate), and the MAGL inhibitors MJN110 (2,5dioxopyrrolidin-1-yl 4-(bis(4-chlorophenyl)methyl)piperazine-1-carboxylate) and JZL184 (4-[Bis(1,3benzodioxol-5-yl)hydroxymethyl]-1-piperidinecarboxylic acid 4-nitrophenyl ester) fully substituted for SA-57. Although the FAAH inhibitors PF-3845 ((N-3-pyridinyl-4-[[3-[[5-(trifluoromethyl)-2pyridinyl]oxy]phenyl]methyl]-1-piperidinecarboxamide) and URB597 (cyclohexylcarbamic acid 3'(aminocarbonyl)-[1,1'-biphenyl]-3-yl ester) did not substitute for SA-57, PF-3845 produced a two-fold leftward shift in the MJN110 substitution dose response curve. In addition, the CB1 receptor antagonist rimonabant blocked the generalization of SA-57 as well as substitution of CP55,940, JZL195, MJN110, and JZL184. These findings suggest MAGL inhibition plays a major role in the CB1 receptor-mediated SA-57 training dose, which is further augmented by FAAH inhibition.
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Introduction Cannabinoid CB1 (Devane et al., 1988; Matsuda et al., 1990) and CB2 receptors (Munro et al.,
1993) and their endogenous ligands N-arachidonoyl ethanolamine (anandamide; AEA) (Devane et al., 1992) and 2-arachidonoylglycerol (2-AG) (Mechoulam et al., 1995; Sugiura et al., 1995) represent primary elements of the endocannabinoid system. This system modulates many physiological processes, including pain (Hohmann et al., 2005; Kinsey et al., 2010; Woodhams et al., 2012; IgnatowskaJankowska et al., 2014), memory (Hampson and Deadwyler, 1999), appetite (Kirkham and Tucci, 2006), and reward (Tsou et al., 1998; Marsicano and Lutz, 1999). The primary psychoactive constituent of Cannabis, Δ9-tetrahydrocannabinol (THC) (Gaoni and Mechoulam 1964) produces its psychotomimetic effects through CB1 receptors (Huestis et al., 2001), and induces dopamine release in the nucleus accumbens (Chen et al., 1991), though to a substantially lower magnitude than other abused drugs. Curiously, THC produces reinforcing effects in some (Gardner et al., 1988; Lepore et al., 1996; Justinova et al., 2003, 2005), but not all (Vlachou et al., 2007; Wiebelhaus et al., 2015) preclinical laboratory animal models. In contrast, THC serves as a reliable discriminative stimulus in the drug discrimination paradigm (Henriksson et al., 1975; Järbe, 1989; Wiley et al., 1997; Vann et al., 2009), an assay that is highly predictive of drug psychoactivity in humans (Chait et al., 1988; Kamien et al., 1993; Lile et al., 2012).
Whereas THC elicits relatively long-lasting pharmacological effects, AEA and 2-AG produce short-lived effects because of rapid hydrolysis by their respective primary catabolic enzymes fatty acid amide hydrolase (FAAH) (Cravatt et al., 1996, 2001) and monoacylglycerol lipase (MAGL) (Di Marzo et al., 1999; Dinh et al., 2002). Accordingly, inhibitors of these enzymes elevate endocannabinoid brain levels, and represent useful investigative tools. Although the selective FAAH inhibitors URB597 (Fu et al., 2005) and PF-3845 (Ahn et al., 2009) elevate AEA brain levels and produce antinociceptive effects, neither compound substitutes for THC (Gobbi et al., 2005; Wiley et al., 2014). Similarly, the MAGL
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inhibitor JZL184 elevates endogenous 2-AG brain levels and produces antinociception, but only partially substitutes for THC (Long et al. 2008; Long et al. 2009; Wiley et al. 2014; Walentiny et al. 2015). Conversely, the dual FAAH-MAGL inhibitor JZL195 fully substitutes for THC, elicits a constellation of cannabimimetic effects (Long et al., 2009; Wise et al., 2012; Hruba et al., 2015) and produces an increased magnitude of antinociceptive effects compared with single enzyme inhibition (Long et al., 2009; Ghosh et al., 2015). Similarly, the dual FAAH-MAGL inhibitor SA-57 fully substitutes for THC in wild-type mice (Hruba et al. 2015).
As it has yet to be established whether an inhibitor of endocannabinoid hydrolysis can serve as the training drug in drug discrimination procedures, the present study investigated whether mice will learn to discriminate SA-57 from vehicle. SA-57 inhibits FAAH much more potently than it inhibits MAGL or ABHD6, another serine hydrolase that degrades 2-AG, but to a much less extent than MAGL (Blankman et al., 2007). Thus, SA-57 possesses utility to investigate the consequences of maximally elevating brain AEA levels, while dose-dependently increasing brain 2-AG levels (Niphakis et al., 2012). To select the SA-57 training dose, initial experiments examined its dose-effect relationship to substitute for the potent CB1 receptor agonist CP55,940 in C57BL/6J mice and AEA in FAAH (-/-) mice (to prevent rapid hydrolysis). Having established that mice learn to discriminate SA-57 from vehicle, we then assessed its dose-response relationship and time course. Because various substrates of FAAH (e.g., AEA, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA)) and MAGL (e.g., 2-AG) bind CB1, CB2, TRPV1(Smart et al., 2000), and peroxisome proliferator-activated receptor-alpha (PPARα) receptors (Lo Verme et al., 2005), we tested whether antagonists for these receptors would block the discriminative stimulus effects of SA-57. Additionally, we conducted an extensive series of drug substitution tests to gain further insight into the training dose of the SA-57 discriminative stimulus. Specifically, we tested whether CP55,940, as well as the non-cannabinoid psychoactive drugs nicotine and diazepam would substitute for the SA-57. As MAGL also plays a rate limiting role in the production
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of arachidonic acid and prostanoids in brain (Nomura et al., 2011), we examined whether the COX-2 inhibitor valdecoxib, which reduces prostanoid synthesis but does not affect brain endocannabinoid levels, would substitute for SA-57. The final goal of the present study was to elucidate the degree to which relevant endocannabinoid hydrolytic enzyme inhibitors contribute to the SA-57 training dose. Accordingly, we investigated whether individual FAAH, MAGL, and ABHD6 inhibitors, we well as simultaneous inhibition of FAAH and MAGL would substitute for SA-57.
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Materials and methods Subjects
Male C57BL6/J mice (Jackson Laboratory; Bar Harbor, ME) and male FAAH (-/-) mice served as subjects. The FAAH (-/-) mice were backcrossed >14 generations on to a C57BL6/J background. The mice were 9-11 weeks of age at the beginning of training and were individually housed in a temperaturecontrolled (20-22°C) vivarium in accordance with Virginia Commonwealth University Institutional Animal Care and Use Committee guidelines. Mice were given water ad libitum, and were food restricted to 85-90% of free-feed body weight, which was established during a two-week period of ad libitum food every six months.
Drugs
SA-57, MJN110, KT182, KT195, and JZL195 were synthesized in the Cravatt laboratory, as previously described (Long et al., 2009; Niphakis et al., 2012, 2013; Hsu et al., 2013). N-arachidonoyl ethanolamine (AEA) was provided by Organix Inc. (Woburn, MA), and valdecoxib was provided by Sigma-Aldrich (Saint Louis, MO). CP55,940, JZL184, PF-3845, rimonabant, and SR144528 were generously supplied by the National Institute on Drug Abuse (NIDA) (Rockville, Maryland, USA). Capsazepine was purchased from Cayman Chemical, and GW6471 was purchased from Tocris Bioscience. Each compound was dissolved in a vehicle consisting of ethanol, emulphor-620 (Rhodia, Cranbury, New Jersey, USA), and saline in a ratio of 1:1:18. All injections were given via the intraperitoneal (i.p.) route of administration in a volume of 10 μl per 1 g of body weight.
Apparatus Drug discrimination was conducted in eight sound-attenuating operant conditioning boxes (18 x 7

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18 x 18 cm) (MED Associates, St. Albans, VT). Each operant box contained two nose poke apertures, and a food dispenser delivering 14-mg food pellets to a receptacle chamber located between apertures. Computer software (MED-PC® IV, MED Associates, St. Albans, VT) was used to record nose pokes and to control stimulus presentations and food deliveries.
Drug Discrimination Paradigm Training
Separate groups of mice were trained to discriminate each of the following three training drugs from vehicle. Groups 1 and 2 consisted of C57BL6/J mice (n=8) trained to discriminate CP55,940, and FAAH (-/-) mice (n=11) trained to discriminate AEA, respectively. The third group of mice consisted of three cohorts of C57BL6/J mice (n=8/cohort) trained to discriminate SA-57 from vehicle. The treatment conditions for each cohort are described below under Testing. The pretreatment times for the training drugs were 120 min for SA-57 and 30 min for CP55,940 and AEA. During each 15 min training session, both nose poke apertures were available, but only responses into the correct aperture associated with the appropriate training drug or vehicle resulted in food reinforcement. Each incorrect response reset the response requirement. Injections before training sessions were conducted (Monday-Friday) in a double alternation sequence of drug (SA-57, CP55,940, or AEA) and vehicle (e.g., vehicle, vehicle, drug, drug).
Testing
Test sessions were scheduled twice per week, with a minimum of 72 h between test days. To be eligible for testing, subjects were required to meet the following three criteria on nine of the previous ten consecutive training sessions: 1) correct completion of the first FR10 (i.e., first 10 consecutive responses into the appropriate aperture); 2) ≥ 80% correct responding; and 3) maintain response rates ≥ 10 responses/min. During the 15-min test sessions, responses in either aperture resulted in the delivery of
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food reinforcement according to an FR10 schedule of reinforcement, without a limitation on the number of reinforcers earned within a session. Before conducting substitution tests, dose-response tests with SA57, CP55,940 or AEA were conducted to characterize their generalization gradients to their respective discriminative stimulus. For time course studies, animals were injected with SA-57 (10 mg/kg) and tested at 0.25, 1, 2, 4, or 8 h after injection. In order to assess whether CB1 receptors mediated the discriminative effects of SA-57, and the substitution of CP55,940, MJN110, JZL184, and JZL195, we used rimonabant (3 mg/kg; Rinaldi-Carmona, 1994). We also examined whether the CB2 receptor antagonist SR144528 (3 mg/kg; Rinaldi-Carmona et al., 1998), the TRPV1 receptor antagonist capsazepine (5 mg/kg; Kinsey et al. 2009), and the PPARα receptor antagonist GW6471 (2 mg/kg; Lo Verme et al. 2005) would block the discriminative stimulus effects of SA-57. Each antagonist was administered 15 min prior to injections of 10 mg/kg SA-57. The three cohorts of mice trained to discriminate SA-57 were employed in the following experiments. All cohorts were included in the SA57 acquisition curve. Cohort 1 was used in the time-course study, the MJN110 (0.25 – 5 mg/kg), KT182 (1 and 2 mg/kg), KT195 (40 mg/kg), valdecoxib (10 mg/kg), and MJN110 (2.5 mg/kg) + PF3845 (10 mg/kg) substitution studies; cohort 2 was used to test the psychoactive non-cannabinoid drugs nicotine (1.5 mg/kg) and diazepam (10 mg/kg), and in substitution tests with JZL195 (2-20 mg/kg), JZL184 (4100 mg/kg), PF3845 (10 and 30 mg/kg), and URB597 (10 mg/kg); and cohort 3 was used in the receptor antagonist experiments (rimonabant, SR144528, capsazepine, GW6471).
[3H]SR141716A binding assay Cerebella were dissected from adult male ICR mice, stored at -80°C, and membranes were
prepared as described previously (Selley et al., 2004). Membrane protein (15 μg) was incubated with 0.94 nM [3H]SR141716A in assay buffer (50 mM Tris-HCl, pH 7.4, 3 mM MgCl2 and 0.2 mM EGTA) with 0.5% (wt/vol) bovine serum albumin (BSA) in the presence and absence of 5 μM unlabeled
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SR141716A to determine non-specific and specific binding, respectively. The assay was incubated for 90 min at 30°C and terminated by rapid filtration under vacuum through Whatman GF/B glass fiber filters that were pre-soaked in Tris buffer containing 0.5% (wt/vol) BSA (Tris-BSA), followed by five washes with cold Tris-BSA. Bound radioactivity was determined by liquid scintillation spectrophotometry at 45% efficiency in ScintiSafe Econo 1 scintillation fluid after a 12-h delay.
Data analysis The percentage of drug appropriate responses and response rates (responses/min) were recorded
for each experiment. Full substitution was defined as greater than or equal to 80% nose pokes that occurred into aperture associated with the training drug. Partial substitution was defined as greater than or equal to 20% and less than 80% nose pokes in the training drug-paired aperture. Less than 20% nose pokes on the drug-paired aperture was defined as no substitution (Solinas et al., 2006). ED50 values (and 95% confidence intervals) for generalization or substitution were calculated using least squares linear regression analysis. Behavioral data are depicted as mean ± S.E.M. The data were analyzed using oneway or two-way ANOVA. Dunnett's tests or Bonferroni post hoc analyses were used following a significant ANOVA for the response rate data. GraphPad Prism 6.0 statistical software (Graph Pad Software, Inc., La Jolla, CA) was used for data analysis.
Binding data were determined in triplicate and are reported as specific binding. Each competition dataset was analyzed by one-way ANOVA to determine concentration-dependence. Rimonabant competition curves were analyzed by non-linear regression to determine IC50 and Hill coefficients using a four parameter fit with GraphPad Prism 6.0. The IC50 values were then converted to Ki values using the Cheng-Prusoff equation.
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