A Novel Nociceptin Receptor Antagonist LY2940094 Inhibits Excessive Feeding Behavior in Rodents: a Possible Mechanism for the Treatment of Binge Eating Disorder

Home , Nociceptin, Nociceptin receptor

1521-0103/356/2/493–502$25.00 http://dx.doi.org/10.1124/jpet.115.228221 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 356:493–502, February 2016 Copyright ª 2016 by The American Society for Pharmacology and Experimental Therapeutics

A Novel Nociceptin Receptor Antagonist LY2940094 Inhibits Excessive Feeding Behavior in Rodents: A Possible Mechanism for the Treatment of Binge Eating Disorder

Michael A. Statnick, Yanyun Chen, Michael Ansonoff, Jeffrey M. Witkin, Linda Rorick-Kehn, Todd M. Suter, Min Song, Charlie Hu, Celia Lafuente, Alma Jiménez, Ana Benito, Nuria Diaz, Maria Angeles Martínez-Grau, Miguel A. Toledo, and John E. Pintar Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana (M.A.S., Y.C., J.M.W., L.R.K., T.M.S., M.S., C.H.); Eli Lilly and Company, Madrid, Spain (C.L., A.J., A.B., N.D., M.A.M.G., M.A.T.); and Rutgers Robert Wood Johnson Medical School, Downloaded from Piscataway, NJ 08854 (M.A., J.E.P.) Received July 31, 2015; accepted December 2, 2015

ABSTRACT

Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide, is of a palatable high-energy diet, reducing caloric intake to control jpet.aspetjournals.org the endogenous ligand of the ORL1/nociceptin-opioid-peptide chow levels. In dietary-induced obese rats, LY2940094 inhibited (NOP) receptor. N/OFQ appears to regulate a variety of physi- feeding and body weight regain induced by a 30% daily caloric ologic functions including stimulating feeding behavior. Recently, restriction. Last, in dietary-induced obese mice, LY2940094 a new class of thienospiro-piperidine-based NOP antagonists was decreased 24-hour intake of a high-energy diet made freely described. One of these molecules, LY2940094 has been identi- available. These are the first data demonstrating that a system- fied as a potent and selective NOP antagonist that exhibited ically administered NOP receptor antagonist can reduce feed- activity in the central nervous system. Herein, we examined the ing behavior and body weight in rodents. Moreover, the

effects of LY2940094 on feeding in a variety of behavioral models. hypophagic effect of LY2940094 is NOP receptor dependent at ASPET Journals on September 23, 2021 Fasting-induced feeding was inhibited by LY2940094 in mice, an and not due to off-target or aversive effects. Thus, LY2940094 effect that was absent in NOP receptor knockout mice. Moreover, may be useful in treating disorders of appetitive behavior such NOP receptor knockout mice exhibited a baseline phenotype as binge eating disorder, food choice, and overeating, which of reduced fasting-induced feeding, relative to wild-type litter- lead to obesity and its associated medical complications and mate controls. In lean rats, LY2940094 inhibited the overconsumption morbidity.

1 Introduction activated-protein kinases, activation of K conductance and inhibition of Ca21 conductance (see reviews by Mogil and Nociceptin/orphanin FQ (N/OFQ) is a 17-amino acid neuro- Pasternak, 2001; New and Wong, 2002). The NOP receptor peptide with high homology to the opioid peptide dynorphin. is widely expressed in the central nervous system (CNS) N/OFQ was identified as the natural ligand for the orphan including the cortex, hippocampus, medial septum, amygdala, receptor ORL1 (today referred to as the NOP receptor) (Meunier thalamus, hypothalamus, and the nucleus of the solitary tract. et al., 1995; Reinscheid et al., 1995). The NOP receptor is a Moreover, NOP is expressed in the peripheral nervous system Class A G-protein-coupled receptor that is functionally cou- as well as in the gastrointestinal tract, smooth muscles, and in pled to inhibition of adenylate cyclase, activation of mitogen- cells of the immune system. Initially, N/OFQ was found to producehyperalgesia,leadingtothenamingthepeptide nociceptin (Meunier et al., 1995); however, in addition to pain, This work was financially supported by Eli Lilly and Company. The authors N/OFQ modulates other CNS behaviors, including stress and Michael A. Statnick, Yanyun Chen, Jeffrey M. Witkin, Linda Rorick-Kehn, Todd M. Suter, Min Song, Charlie Hu, Celia Lafuente, Alma Jiménez, Ana anxiety, depression, substance abuse, prolactin secretion, Benito, Nuria Diaz, Maria Angeles Martínez-Grau, and Miguel A. Toledo are locomotor activity, body temperature, memory, and feeding employees and shareholders of Eli Lilly and Company. ’ M.A.S. and Y.C. contributed equally to the manuscript. (Mustazza and Bastanzio, 2011; Gavioli and Calo , 2013; Singh dx.doi.org/10.1124/jpet.115.228221. et al., 2013; Witkin et al., 2014).

ABBREVIATIONS: ANOVA, analysis of variance; BED, binge eating disorder; CHO, Chinese hamster ovary; CNS, central nervous system; DIO, dietary induced obese; HED, high-energy diet (diet high in both fat and sucrose); 5-HT, 5-hydroxytryptamine or serotonin; J113397, 1-[(3R,4R)-1-cyclooctylmethyl-3- hydroxymethyl-4-piperidyl]-3-ethyl-1, 3-dihydro-2H-benzimidazol-2-one; JTC-801, N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamidehy- drochloride; KO, knockout; LY2940094, [2-[4-[(2-chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4’-piperidine]-1’-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl] methanol; mCPP, meta-chlorophenylpiperazine; N/OFQ, nociceptin/orphinan FQ (F:phenylalanine; Q, glutamine; the amino acids that begin and end the peptide sequence); NOP, nociceptin opioid peptide receptor; ORL1, opioid-receptor-like-1; RO, receptor occupancy; Ro 64-6198, (1S,3aS)-8- (2,3,3a,4,5,6- hexahydro-1H-phenalen-1-yl)-1-phenyl-1,3,8-triaza- spiro[4.5]decan-4-one; RQ, respiratory quotient; SB-612111, [(2)-cis-1-methyl-7-[[4-(2,6-dichlorophenyl) piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5H-benzocyclohepten-5-ol; VO2, oxygen consumption; WT, wild type.

493 494 Statnick et al.

Numerous studies have reported that activation of NOP Perkin-Elmer Life and Analytical Sciences (Waltham, MA). A 20% receptors produces a robust orexigenic effect in rodents. Captisol in 25 mM PO4 buffer (pH 2) formulation was used in the Indeed, fasting regulates N/OFQ and NOP receptor gene studies, and all doses were corrected for the salt weight. This expression in the CNS (Rodi et al., 2002; Przydzial et al., formulation permitted the compound to be completely dissolved in 2010). Highly potent and efficacious orexigenic effects are solution. produced by administration of either N/OFQ peptide analogs or small molecule agonists (Pomonis et al., 1996; Polidori Animals et al., 2000), leading the authors to hypothesize that the Male lean and DIO Long-Evans rats were purchased from Harlan feeding effects of N/OFQ are mediated at the level of the Sprague-Dawley (Indianapolis, IN) and male DIO C57Bl/6 mice were forebrain and/or hypothalamus (Polidori et al., 2000). Micro- purchased from Taconic (Hudson, NY). Male NOP receptor knockout injection studies confirmed this hypothesis and identified the (KO) mice and wild-type littermates maintained on a 129S6 inbred nucleus accumbens and ventromedial hypothalamic nucleus background were generated by heterozygous breeding as previously described (Kest et al., 2001). All animals were singly housed in rooms as specific sites of the orexigenic actions of N/OFQ (Stratford using a 12-hour light/dark cycle and had ad libitum access to food and et al., 1997). It is postulated that the orexigenic effects of water unless otherwise stated. Lean animals were maintained on N/OFQ are the result of inhibition of anorexigenic signaling chow diet TD2014, whereas DIO animals were maintained on the (Farhang et al., 2010). These data support the hypothesis that high-energy diet TD95217 (Harlan Teklad, Madison, WI). All in vivo inhibitors to NOP receptor signaling will regulate food intake experiments were performed during the “lights on” photoperiod unless and might therefore be useful in the treatment of obesity and otherwise stated. Animals were maintained and experiments were Downloaded from eating disorders. conducted in compliance with the policies of the Animal Care and Use Several NOP selective antagonists (J113397, JTC-801, and Committee of Eli Lilly and Company in conjunction with the American SB-612111) and agonists (Ro 64-6198 and W212393) have Association for the Accreditation of Laboratory Animal Care-approved been developed (Zaveri et al., 2005; Largent-Milnes and guidelines. Vanderah, 2010). Recently, we identified a novel series of NOP receptor antagonists based on a dihydrospiro(piperidine- In Vitro NOP Receptor Binding jpet.aspetjournals.org 4,7’thieno[2,3-c]pyran) scaffold (Toledo et al., 2014). These A filtration-based [3H]-nociceptin binding assay was used to de- antagonists were found to have high affinity and antagonist termine the affinity (Ki) of LY2940094 using previous conditions with activity for the human NOP receptor. In vivo, we found that minor modifications (Ardati et al., 1997). Assay incubations were 3 the antagonists had good oral bioavailability and exhibited performed in deep-well 96-well plates with [ H]-nociceptin (final assay – CNS activity. One of these molecules, LY2940094 (Toledo concentration 0.2 nM) and 5 10 mg of membrane protein (isolated from CHO cells expressing cloned human NOP receptors) in a final volume et al., 2014), exhibited subnanomolar antagonist potency (Kb 5 of 0.5 ml of HEPES buffer (20 mM; pH 7.4) containing, 5 mM MgCl2, at ASPET Journals on September 23, 2021 0.17 nM) in vitro at the human NOP receptor. Moreover, 1 mM EDTA, 100 mM NaCl, and 0.1% bovine serum albumin and 5 5 LY2940094 was orally bioavailable in rats (F 57%, T1/2 3.8 incubated for 60 minutes at 25°C. Reactions were terminated by hours) in vivo and demonstrated sustained NOP receptor filtration on glass fiber filtermats GF/C Filtermat A (Perkin-Elmer occupancy in the brain p24 hours receptor occupancy (RO) 5 Life and Analytical Sciences) pretreated with 0.3% polyethyleneimine. 62% at 10 mg/kg orally]). When tested in vivo LY2940094 The filters were washed three times with 5 ml of ice-cold Tris·HCl potently inhibited the hypothermic effects of exogenously buffer (50 mM, pH 7.4) and then dried and embedded with MeltiLex administered NOP agonist Ro 64,6198, confirming its on- scintillant (Perkin-Elmer Life and Analytical Sciences), and the bound target activity. radioactivity was counted in a Microbeta Trilux (Perkin-Elmer Life Here we hypothesize that inhibition of NOP receptor and Analytical Sciences). Specific binding was determined by displacement with 100 nM unlabeled nociceptin. Curves were plotted as signaling in the CNS will modulate feeding behavior. We the percent of specific inhibition versus LY2940094 concentration, demonstrate for the first time that a small molecule, orally and IC50 values were determined using four-parameter nonlinear bioavailable, CNS-penetrant NOP receptor antagonist, regression (XLFit version 4.0, IDBS). Ki values were calculated from LY2940094, does indeed inhibit feeding behavior in rats and the IC50 according to Cheng and Prusoff, where Ki 5 IC50 Â (1 1 D Â 21 mice in a genotype-specific manner and stimulates lipid Kd–1) (Cheng and Prusoff, 1973). Reported values for Ki are shown utilization in diet-induced obese mice. as geometric means 6 the standard error of the mean (S.E.M.), from n 5 3 independent assays. Geometric means are calculated by the

equation GeoMean 5 10[average (log Ki1 1 log Ki2 1...log KIn)/square root of the number of replicates, n]. Materials and Methods Compounds, Dose Selection, and Formulation In Vitro Opioid Receptor Binding [2-[4-[(2-Chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4’-piperidine]- To determine receptor selectivity to classic opioid receptors, the

1’-yl)methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol (LY2940094) as a binding affinity (Ki) of LY2940094 was determined in CHO or human free base or a tartrate salt was used for all studies and was synthesized embryonic kidney cell membranes expressing cloned human m, k, at Lilly Research Laboratories (Indianapolis, IN) as previously de- or d receptors, as previously described (Emmerson et al., 2004). scribed (Toledo et al., 2014). Doses of LY2940094 were selected that Briefly, membranes (4–6 mg protein per reaction) were added to buffer were previously shown to produce saturating in vivo occupancy of containing 50 mM Tris-HCl, 100 mM NaCl, 1 mM GDP, 5 mM MgCl2, NOP receptors in the rat hypothalamus for at least 6 hours to and 1 mM EDTA (pH 7.4). The reactions were initiated by the addition ensure suitable receptor blockade over the testing period (Toledo of 0.2 nM [3H]-diprenorphine to yield a 500 ml final assay volume. The et al., 2014; Barth et al., 2014). The serotonin 2C receptor agonist reaction was incubated for 120 minutes at 25°C. Specific binding was meta-chlorophenylpiperazine (mCPP) was used at an ED90 dose as determined by displacement with 10 mM naltrexone. Reactions were a positive control for the NOP receptor knockout mouse study and was terminated by rapid filtration through glass fiber filters, radioactivity purchased from Sigma-Aldrich (St. Louis, MO). Naltrexone HCl was was measured, and data were analyzed using procedures identical to purchased from Sigma-Aldrich. [3H]-nociceptin was purchased from those used for NOP receptor binding. LY2940094 Inhibits Food Intake in Rodents 495

In Vitro Functional Activity on G-protein Activation Hyperphagia Stimulated by Consumption of a Highly Palatable Diet in Lean Rats. Male Long-Evans rats (376–445 g) The NOP receptor antagonist affinity (K ) of LY2940094 was b at 9–10 weeks of age were individual housed and accustomed to a measured in CHO membranes expressing cloned human NOP recep- 12-hour light/dark cycle on a reverse light cycle (lights off at 10:00 AM) tors with a GTPg-[35S] binding assay, according to previously de- for at least 1 week before the start of experiment. All rats were scribed protocols with minor modifications (Delapp et al., 1999; Ozaki maintained on a standard chow diet TD2014 (Harlan) ad libitum with et al., 2000). Assays were in a 200 ml volume with a 20 mM HEPES free access to water. Before receiving drug treatment, a mean daily buffer containing 100 mM NaCl, 5 mM MgCl , 1 mM EDTA, 0.1% 2 food intake was obtained for 3 days for each rat by dosing all rats with bovine serum albumin, 3 mM GDP, 0.5 nM GTPg[35S]. NOP receptor vehicle. Rats were divided into four treatment groups by block membrane suspension was added at a concentration of 20 mg pro- randomization according their average food intake (n 5 7 rats per tein per well, and receptor stimulation was achieved with 300 nM group). On the day of study, rats were treated with either LY2940094 nociceptin. Wheat germ agglutinin-coated scintillation proximity (10 or 30 mg/kg) or vehicle by oral gavage. These doses of LY2940094 assay beads (Perkin-Elmer Life and Analytical Sciences) were added are known to saturate NOP receptors in the hypothalamus in vivo at 1 mg per well to detect membrane-bound GTPg[35S]. Plates were while maintaining target selectivity based on our study in NOP sealed and incubated for 2 hours at 25°C and then placed at 4°C receptor KO mice (see above). Thirty minutes after drug administra- overnight to allow the scintillation proximity assay beads to settle. tion, rats in three treatment groups (vehicle, 10 mg/kg, 30 mg/kg) were Plates were then counted for radioactivity in a Microbeta Trilux given access to a high-energy diet (HED) TD95217 (Harlan Teklad), instrument. Specific GTPg[35S] binding was determined as the whereas one group (vehicle treated) was maintained on the standard difference in counts per minute observed in the absence and presence

rat chow diet to measure the amount of hyperphagia stimulated by Downloaded from of 10 mM unlabeled GTPgS. Data were plotted as the percent of access to the HED. Food intake was monitored for 5 hours after access specific GTPg[35S] bound from which IC values were determined 50 to the HED. Three-day cumulative food intake and body weight were using four-parameter nonlinear regression routines (XLFit version also measured after daily treatment with LY2940094. 4.0, IDBS). Antagonist affinity (K ) was estimated according to Delapp b Hyperphagia Stimulated by a Mild Calorie Restriction Diet et al. (1999) using a modification of the equation of Cheng and Prusoff 2 in DIO Rats. Overeating commonly occurs when a person is dieting (1973) where K 5 IC Â (1 1 D Â EC –1) 1. Reported values for K b 50 50 b or immediately after cessation of the diet. To test how NOP receptor are shown as geometric mean 6 the standard error of the mean (S.E.M.)

blockade would function in the context of dieting, male DIO Long jpet.aspetjournals.org from three independent experiments. Evans rats (520–631 g) at 14–15 weeks of age were placed under a mild Agonist activity was assessed using a functional GTPg[35S] binding caloric restriction designed to model the reduced calorie intake that assay similar to those described for antagonist measurement, except occurs when obese people diet (generally a 20–30% reduction in daily that the agonist assay did not have added nociceptin to stimulate caloric intake). All rats were fed the HED TD9521 for 10–12 weeks NOP receptor-mediated G-protein activation. Basal activity was before the start of the study for them to develop obesity. The rats were determined in the absence of added nociceptin. A control nociceptin individually housed and accustomed to 12-hour light/dark on a reverse concentration response curve (geometric mean EC for nociceptin was 50 light cycle (lights off at 10:00 AM) at 24°C for at least 1 week before the 2.25 nM, S.E.M. 5 0.41, n 5 18) was included in each assay. The start of the experiment. All rats were maintained on HED TD95217 at ASPET Journals on September 23, 2021 concentration range tested for LY2940094 in the agonist assay was (Harlan Teklad) with free access to water throughout the study. Before 0.17 nM to 10 mM (final assay dimethylsulfoxide concentration in initiating the mild caloric restriction, the rats were dosed orally with all wells was 1%). The percent efficacy was calculated relative to vehicle, and food intake and body weight were monitored for 3 days so nociceptin, which was set at 100%. that average food intake was obtained for each rat. Rats were then grouped by block randomization according their body weight into 5 Models of Hyperphagia groups (n 5 6 rats/group). During the experiment rats were treated Fasting-Induced Food Intake Study in Mice. Experiments orally with either vehicle or LY2940094 (10 mg/kg, a dose previously were performed on 12-week-old male NOP receptor knockout mice and shown to inhibit food intake in rats) 30 minutes before the start of the wild-type (WT) littermates maintained on a 129S6 inbred background. dark cycle. Group A rats were treated with vehicle and fed ad libitum Individually housed mice were acclimated to housing conditions for a with TD 95217 diet throughout the entire 4-day study period. Groups minimum of 3 days before the onset of testing to eliminate stress B and C were treated with vehicle and fed with 70% of their average effects due to the change from group to individual housing. Mice of daily food intake for 3 days, whereas Groups D and E were treated each genotype (n 5 3 per treatment group per test day) were randomly with LY2940094 (10 mg/kg orally) and fed with 70% of their average assigned to each treatment group on each test day. Food was removed from cages just before the onset of the dark cycle. After a 15-hour fast, mice were administered LY2940094 at doses (3 or 30 mg/kg orally) that produce saturating levels of NOP receptor occupancy in the hypothalamus (Barth et al., 2014) or vehicle via oral gavage 1 hour before gaining ad libitum access to food during the “lights on” period

(LabDiet 5058). An additional group of mice received the 5-HT2C receptor agonist mCPP (at an ED90 dose of 30 mg/kg, i.p.), which is known to potently inhibit food intake to serve as a positive control for both genotype (to confirm that NOP receptor knockout mice do not exhibit an abnormal feeding response to a known anorectic agent) and compound treatment. Measurements of food intake were recorded 1 hour after access to food during the light phase, a time during which mice are typically at rest and not normally eating. The first hour after food presentation represents a period of the most robust hyperphagia, after which the animals eat at a much slower normal rate. After initial testing, mice were rested for 1 week with ad libitum access to chow. After the week of rest, mice were retested after a Latin-square study design. Therefore, at the end of the study all mice received all drug Fig. 1. Chemical structure of the nociceptin receptor antagonist [2-[4-[(2- (LY2940094 and mCPP) and vehicle treatments; thus each treatment chloro-4,4-difluoro-spiro[5H-thieno[2,3-c]pyran-7,4’-piperidine]-1’-yl) group at the end of the study had an n 5 12. methyl]-3-methyl-pyrazol-1-yl]-3-pyridyl]methanol (LY2940094). 496 Statnick et al. daily food intake for 3 days. On day 4, the forced caloric restriction was with LY2940094 was measured and analyzed by one-way ANOVA lifted, and all the groups of rats were returned to ad libitum feeding with a Tukey post hoc test, whereas cumulative daily HED intake was conditions. During day 4 ad libitum feeding, Groups B and D were analyzed using a two-way ANOVA with time and drug treatment as treated with vehicle, whereas Groups C and E were treated with factors followed by a Bonferroni’s multiple comparison post hoc test. In LY2940094 (10 mg/kg orally). Daily food intake and body weight were calorie-restricted DIO rats, changes in food intake and body weight monitored for the entire period of the study. Six-hour and 24-hour food after treatment with LY2940094 were analyzed using a one-way intake and body weight change after last dosing (day 4) were also ANOVA with Tukey post hoc test. In ad libitum-fed DIO mice, food measured. Body weight was not different between the groups on day 4, intake was measured after LY2940094 treatment in time bins (0- to 2-, therefore, the groups were separated into days 1–3 (the period during 2- to 4-, 4- to 6-, and 6- to 24-hour bins) and analyzed by two-way the restricted feeding) and day 4 (when all animals were returned to ANOVA with time and drug treatment as factors followed by a ad libitum feeding conditions). Bonferroni’s multiple comparison post hoc test, whereas changes in body weight were analyzed by a Student’s t test. Model of Ad Libitum Feeding and Energy Utilization Free Access Food Intake in DIO Mice. To determine if Results blockade of NOP receptors was effective in reducing free access feeding in the context of obesity we conducted a study in DIO mice. LY2940094 Is a Potent and Selective Antagonist at NOP Three- to four-month-old male diet-induced obese (DIO) C57Bl/6 Receptors mice (40.3–44.1 g) were purchased from Taconic (Germantown, LY2940094 (Fig. 1) exhibits high affinity binding, high Downloaded from NY). Animals were individually housed in a temperature-controlled (24°C) facility with a 12-hour light/dark cycle (lights on 10:00 PM to functional antagonist potency with no agonist activity, and facilitate animal dosing just before the start of the dark cycle) and had high selectivity against other opioid receptors (Tables 1 and 2). free access to a HED (Teklad TD95217) and water. Before the experiment The binding affinity for LY2940094 in membranes expressing started, mice were treated with vehicle by oral gavage and weighed for recombinant human NOP receptors was Ki 5 0.1 nM. High 2 days to acclimatize them to the handling procedures. The day before binding affinity was found in membranes isolated from rat 5 the study, mice were block randomized by body weight to treatment brain, Ki 0.7 nM, confirming that LY2940094 is potent at jpet.aspetjournals.org groups (n 5 8/group) so that each group had mean body weight that did both the rodent and human NOP receptors. Antagonist not significantly differ. potency of LY2940094 was similarly high at the human NOP Study 1: The effect of LY2940094 on food intake and body weight in receptor, K 5 0.17 nM. No agonist activity of LY2940094 at DIO mice. Just before the onset of the dark photoperiod, LY2940094 b human NOP receptors was detected at concentrations up to or vehicle was administered by oral gavage at 20 mg/kg (8 ml/kg) to DIO C57Bl/6 mice. This dose was chosen as doses within this range 10 mM. Moreover, selectivity of LY2940094 over the other classic reduced fasting-induced food intake in mice while maintaining on opioid receptors (mu, delta, and kappa) was greater than 1000-

target specificity (see Results). Food intake was measured at 0- to 2-, 2- fold (Table 2). at ASPET Journals on September 23, 2021 to 4-, 4- to 6-, and 6- to 24-hour bins posttreatment with LY2940094. Animals received a second dose of LY2940094 treatment 24 hours LY2940094 Inhibits Fasting-Induced Feeding in Mice later, and body weight was measured. Study 2: The effect of LY2940094 on energy expenditure and fuel We employed congenic NOP receptor knockout mice on a utilization in DIO mice. Indirect calorimetry was performed to 129/S6 background to examine the effects of LY2940094 on examine the effect of LY2940094 on energy expenditure and fuel fasting-induced feeding and to evaluate whether the effects utilization. After 24 hours of acclimation to the 11 cm Â 21 cm were NOP receptor mediated and not the result of off-target or calorimetry chambers (Columbus Instruments International Corp., toxic effects. We found that both genotype and treatment with 5 Columbus, OH), LY2940094 or vehicle was administered to mice (N LY2940094 significantly reduced fasting-induced food intake 5/group) at 20 mg/kg (8 ml/kg) by oral gavage. Oxygen consumption (VO ) 2 [overall two-way ANOVA for interaction F(3, 88) 5 4.0, P 5 and CO production were measured to calculate the respiratory quotient 2 0.01; genotype F(1, 88) 5 7.1, P 5 0.009; and treatment F(3, (RQ) and energy expenditure. RQ was calculated as the ratio of CO2 88) 5 34, P , 0.001]. Interestingly in vehicle-treated mice, production to VO2. Energy expenditure was calculated as the product of caloric value (CV) and VO2 per kilogram (kg) of body weight, where caloric NOP KO mice consumed less food than WT littermate controls value 5 3.815 1 1.232*RQ (Elia and Livesey, 1992). Locomotor activity (Fig. 2). The reduction in fasting-induced feeding in NOP KO was monitored over 24 hours while the animals were in the calorimetry mice was robust, approximating a 41% reduction from controls chamber by assessing laser beam breaks in the X-, Y-, and Z-planes. (P , 0.01 WT vehicle versus KO vehicle). After the administration of LY2940094 (at doses that produce full RO) to WT Statistical Analysis mice, fasting-induced feeding was significantly inhibited (P , , Data were expressed as mean 6 standard error (S.E.M.). Fasting- 0.005 WT vehicle versus WT at 3 mg/kg and P 0.05 WT induced feeding in mice was analyzed using a two-way analysis of vehicle versus 30 mg/kg LY2940094). Contrary to the response variance (ANOVA) with genotype and drug treatment as factors in WT mice, LY2940094 did not affect feeding in NOP KO mice followed with a Bonferroni’s multiple comparison post hoc test. The at either 3 or 30 mg/kg, doses that decreased feeding in WT amount of HED consumed over 5 hours by lean rats after treatment littermates. To ensure that the NOP KO mice were capable of

TABLE 1 In vitro pharmacology of the NOP receptor antagonist LY2940094 at human (hNOP) or rat (rNOP) receptor expressed in CHO cells

hNOP Binding rNOP Binding Affinity hNOP Antagonist hNOP Agonist Activity Affinity Ki 6 S.E.M., n =3 Ki 6 S.E.M., n =3 Affinity Kb 6 S.E.M., n =3 Emax @10mM, n =3

nM 0.105 6 0.0361 0.71 6 0.1 0.166 6 0.0352 20.500 6 2.52 LY2940094 Inhibits Food Intake in Rodents 497

TABLE 2 and body weight [overall two-way ANOVA was significant for Selectivity of the NOP antagonist LY2940094 at human opioid interaction F(9, 80) 5 2.329, P 5 0.02; time F(3, 80) 5 38.96, receptors P , 0.0001; and treatment F(3, 80) 5 14.6, P , 0.0001]. Three-

Target Assay Ki day cumulative food intake was lower over the testing period

nM in animals treated with LY2940094 compared with vehicle- treated rats on HED alone (P , 0.01) (Fig. 3B). Vehicle-treated Nociceptin Receptor 0.105 Mu Opioid .451 animals eating HED gained a significantly greater amount of Kappa Opioid .430 weight than the chow-fed animals over the testing period (P , Delta Opioid .479 0.0001), whereas LY2940094-treated rats gained less weight (Fig. 3C) (P , 0.01 at 30 mg/kg dose). These data support that the N/OFQ system may be involved in macronutrient prefer- exhibiting a reduction in food intake after the administration ence or palatable food consumption. Moreover, our findings of an anorexigenic agent, we tested the 5-HT2C agonist mCPP. support that this form of hyperphagia, and subsequent body We found that 30 mg/kg mCPP produced a maximum and weight gain can be inhibited by LY2940094. equivalent inhibition of 1-hour food intake in both WT and NOP KO mice (P , 0.001 WT vehicle versus WT and KO). LY2940094 Inhibits Food Intake in Calorie-Restricted DIO Rats. Downloaded from LY2940094 Inhibits Consumption of Palatable Food Intake To model dieting we placed DIO rats on a mild calorie in Lean Rats. restriction (animals were given access to 70% of free access Another model of hyperphagia that we tested was feeding eating) for 3 days. During the 3-day restriction period, animals induced by the acute access to a highly palatable, high energy in Groups B and C received vehicle treatment, whereas diet (HED). In this model lean rats given acute access to a animals in Groups D and E received 10 mg/kg oral LY2940094 HED will over consume the diet preferentially over chow, (Fig. 4). On day 4, Groups A, B, and D received vehicle and jpet.aspetjournals.org ingesting significantly more calories than animals given Groups C and E were treated with 10 mg/kg oral LY2940094. access to chow alone. We found that LY2940094 potently Immediately after the oral treatment on day 4, animals were inhibited 5-hour HED-induced hyperphagia [overall one-way returned to ad libitum food access. Figure 4A shows that in the ANOVA treatment F(3,20) 5 5.536, P 5 0.006]. As can be seen first 6 hours after replenishment of food, calorie-restricted in Fig. 3A, access to a HED resulted in an approximately 36% animals in Groups B and C significantly over consumed the increase in acute caloric intake over rats given access to chow diet compared with the vehicle-treated ad libitum-fed group alone (see vehicle-treated HED versus vehicle-treated chow A [overall one-way ANOVA F(4, 25) 5 4.009, P , 0.05]. groups). Interestingly when given a choice between chow and Continued treatment with LY2940094 (Group E) during at ASPET Journals on September 23, 2021 HED the rats will preferentially eat almost all of their calories restriction and ad libitum periods completely abolished this from the HED and not consume the chow diet. Doses of 10 and caloric restriction-induced hyperphagia. Moreover, adminis- 30 mg/kg of LY2940094 (doses that produce approximately tration of LY2940094 during restriction days and/or on the 100% occupancy) produced a robust inhibition of caloric intake ad libitum day prevented animals from overeating in the of the HED (P , 0.05). Interestingly, the reduction in caloric 24-hours upon food replenishment [Fig. 4B; overall one-way intake approximates that of the chow-only vehicle-treated ANOVA F(4, 25) 5 2.937, P , 0.05]. Examination of changes in animals. Thus, LY2940094 was able to completely prevent body weight during this study revealed that during the calorie the hyperphagia induced by acute access to a HED. When restriction phase of the study (days 1–3) animals in all treatment administered for 3 days, LY2940094 reduced both cumulative groups lost body weight [Fig. 4C; overall one-way ANOVA food intake [overall two-way ANOVA was significant for F(4, 25) 5 7.742, P , 0.001]. Interestingly, animals treated interaction F(9, 80) 5 7.709, P , 0.0001; time F(3, 80) 5 with LY2940094 tended to lose less weight than the vehicle-treated 1127, P , 0.0001 and treatment F(3, 80) 5 44.9, P , 0.0001] controls. However, when the animals were returned to ad libitum

Fig. 2. LY2940094 inhibits fasting-induced food intake in male wild-type 129S6 mice (solid bars) but not NOP receptor knockout mice (open bars) after oral administration. Mice were fasted overnight, dosed with LY2940094 30 minutes before presentation of food during the lights-on period. Food intake was measured for 1 hour. Data were compared with a two-way ANOVA with genotype and treatment as factors followed by Bonferroni’s post hoc test. *P , 0.001 WT vehicle versus ORL KO vehicle; #P , 0.05 WT vehicle versus WT 3 and 30 mg/kg LY2940094; ^P , 0.001 WT vehicle versus WT and ORL1 knockout mCPP, 30 mg/kg. 498 Statnick et al. Downloaded from jpet.aspetjournals.org

Fig. 3. LY2940094 inhibits palatable food intake in lean male Long-Evans rats after oral administration. (A) 5-hour food intake was measured after the acute presentation of a high- energy diet during the lights-on period (**P , 0.01 one-way ANOVA with Tukey’s post hoc test). (B) 24-hour cumulative food intake measured over 3 days. (C) 24-hour cumulative body weight change measured over 3 days. **P , 0.01, ****P , 0.001 two-way ANOVA with Bonferroni’s test. at ASPET Journals on September 23, 2021

feeding conditions on day 4 (when the forced diet was LY2940094 Inhibits 24-hour Free Access Food Intake and lifted) animals treated with LY2940094 gained less weight Stimulates Lipid Utilization in DIO mice than vehicle-treated controls (P , 0.05) [Fig. 4D; overall one- The effect of LY2940094 on spontaneous 24-hour free-access way ANOVA F(4, 25) 5 5.783, P 5 0.002]. Thus, LY2940094 is food intake was measured in DIO C57Bl/6 mice maintained on effective in preventing hyperphagia and weight regain after a a HED. Consistent with the previous findings, LY2940094 period of mild caloric restriction resembling dieting. inhibited spontaneous free-access feeding in DIO mice. To LY2940094 Inhibits Food Intake in Rodents 499

map the time course of the effects of LY2940094 over 24 hours, we measured food intake in 2-hour bins at 20 mg/kg oral dose of LY2940094. This was a dose expected to lack off- target effects based on our knockout mouse study and yet it is highenough to expect exposure to cover 24 hours based upon previous receptor occupancy data (Toledo et al., 2014). We found that 24-hour food intake was reduced [overall two- way ANOVA time F(3, 56) 5 20.51, P , 0.0001; treatment F(1, 56) 5 13.35, P , 0.001, although there was no interaction F(3,56) 5 2.530, P , 0.07] in each of the time bins, reaching statistical significance in the 6- to 24-hour bin (P , 0.001) by 20 mg/kg LY2940094 (Fig. 5A). Cumulative 24-hour intake was significantly reduced [overall two-way ANOVA interaction F(4, 70) 5 5.647, P 5 0.0005; time F(4, 70) 5 60.23, P , 0.0001; treatment F(1,70) 5 30.18, P , 0.0001] by approximately 45% (Fig. 5B). Moreover, body weight was significantly reduced by treatment with LY2940094 (Fig. 5C, P 5 0.0217). Consistent with the reduced food intake, the respiratory quotient (RQ) Downloaded from was significantly reduced by 20 mg/kg LY2940094, indicating a shift from carbohydrate to oxidation of fat to drive metabolism (Fig. 6). LY2940094 at this dose did not affect either energy expenditure or 24-hour locomotor activity (data not shown). jpet.aspetjournals.org Discussion In the present study we tested the hypothesis that a potent and selective NOP receptor antagonist, LY2940094, would reduce overeating and consequently weight gain. We recently reported that LY2940094 occupies NOP receptors for at least 6 hours after oral administration (Toledo et al., 2014) and has at ASPET Journals on September 23, 2021 low intrinsic plasma clearance, making it a good candidate to assess behavior over 24 hours. At doses that fully occupy NOP receptors, LY2940094 inhibited feeding behavior in a variety of rodent models. Critically, we demonstrated that the effects of LY2940094 are due to specific blockade of NOP receptors. NOP receptors are widely distributed in the rat brain. High receptor density has been identified throughout the rat CNS including the hypothalamus (Florin et al., 2000; Gehlert et al., 2006). Recently, we used a liquid chromatography-tandem mass spectrometry method (Pedregal et al., 2012; Toledo et al., 2014) to measure RO of novel NOP receptor antagonists in the hypothalamus of rats. After oral administration, LY2940094 potently occupied NOP receptors in the hypothalamus with an ED80 of 3 mg/kg (E. Raddad et al., submitted manuscript). The duration of RO was reasonably long at a 10 mg/kg dose of LY2940094, providing .80% receptor coverage for at least 4 hours. Therefore, to adequately evaluate the efficacy of LY2940094 we chose doses for our feeding efficacy studies based on doses that would completely saturate NOP receptors. Using the RO data, we chose doses ranging from 3 to 30 mg/kg for our in vivo feeding studies. The effects of N/OFQ on feeding are well documented (for review, see Witkin et al., 2014). Hyperphagia is consistently observed after intracerebroventricular administration of Fig. 4. LY2940094 inhibits food intake and body weight regain induced by N/OFQ into either the lateral or third ventricle but not the 3 days of a mild calorie restriction (30%) in male DIO rats. On days 1–3 fourth ventricle (Pomonis et al., 1996; Polidori et al., 2000). The rats were either fed ad libitum (Group A) or were restricted to 70% of their free feeding (Groups B, C, D, E), during which time animals received either vehicle (Groups A, B, C) or 20 mg/kg oral LY2940094 (Groups D and E). On day 4 all groups were returned to ad libitum feeding, and food intake was monitored after either vehicle (Groups A, B, D) or 20 mg/kg oral weight change, (D) Day 4 body weight change. *P , 0.05 versus Group A, LY2940094 (Groups C and E). (A) Day 4 6-hour cumulative food intake, (B) , , , – **P 0.01 versus Group A ***P 0.001 versus Group A, #P 0.05 versus Day 4 24-hour cumulative food intake, (C) Day 1 3 cumulative body Group E one-way ANOVA with Tukey’s post hoc analysis. 500 Statnick et al.

Fig. 6. LY2940094 decreases RQ in male DIO C57Bl/6 mice maintained on a high-energy diet. Mice were treated with vehicle (d) or 20 mg/kg oral LY2940094 (n) 30 minutes before the onset of the dark cycle. Downloaded from

protein 1 expression after N/OFQ infusion. These data support that N/OFQ effects on energy metabolism engages mechanisms other than stimulating food intake alone. Indeed, genetic deletion of NOP was reported to increase core body temperature in mice, jpet.aspetjournals.org supporting direct tonic actions of endogenous N/OFQ signaling on energy expenditure (Uezu et al., 2004). Interestingly, N/OFQ inhibits activation of anorexigenic proopiomelanocor- tin neurons in association with meal termination (Bomberg et al., 2006; Farhang et al., 2010). Moreover, both N/OFQ and the small molecule NOP agonist Ro 64-6198 inhibit CRF-induced

anorexia (Ciccocioppo et al., 2002, 2004). A working hypoth- at ASPET Journals on September 23, 2021 esis is that N/OFQ may stimulate feeding via inhibition of anorexigenic pathways, resulting in disinhibition of orexigenic mechanisms. Our data would support this hypothesis in that we found that LY2940094 primarily worked in models where the rodent exhibited hyperphagia, stimulated by frank fasting, mild caloric restriction, and weight loss (to mimic dieting), or after the acute presentation of a palatable high-energy diet. Thus a NOP antagonist such as LY2940094 may be most useful in the treatment of disorders associated with hyperphagia, such as binge eating disorder (BED). Recently, Micioni di Bonaventura et al. (2013) reported in a rat model of stress-induced binge eating in animals with a history of calorie restriction, increased the sensitivity to the hyperphagic effects of N/OFQ. Consistent with these findings Fig. 5. LY2940094 inhibits food intake in male DIO C57Bl/6 mice we found that blockade of NOP with LY2940094 was effective maintained on a high-energy diet after oral administration. Mice were treated with vehicle (closed bars or d) or 30 mg/kg oral LY2940094 in reducing hyperphagia associated with mild calorie restric- (hatched bar or n) 30 minutes before the onset of the dark cycle. Food tion as well as fasting. Moreover, the ability of LY2940094 to intake in 2-hour bins (A) and cumulative food intake (B) were measured. inhibit fasting-induced feeding was totally blocked in NOP , , **P 0.05 versus vehicle, ****P 0.0001 versus vehicle by two-way receptor knockout mice, whereas the hypophagic effect of the ANOVA with Bonferroni’s post hoc test. (C) 24-hour body weight change. *P , 0.05 Student’s t test 5HT2C agonist mCPP was preserved in NOP receptor knockout mice. These data demonstrate that 1) genetic deletion of NOP receptors reduces fasting-induced feeding, 2) LY2940094 orexigenic effects of N/OFQ are speculated to arise in the inhibits fasting-induced feeding in WT mice, 3) the anorexi- hypothalamus, a brain region involved in regulating food genic effect of LY2940094 is dependent on NOP receptor intake and that expresses abundant NOP receptors (Stratford expression, 4) the effects of LY2940094 were not the result of et al., 1997). Moreover, chronic administration of N/OFQ by producing aversive actions or other side effects influencing minipump stimulated food intake, decreased energy expendi- feeding, and 5) NOP receptor knockout mice are capable of ture, and produced an increase in body weight gain in mice responding maximally to other anorectic agents such as a (Matsushita et al., 2009). Interestingly, when controlled for 5HT2C agonist. We found a similar effect to inhibit fasting- food intake, N/OFQ infusion still stimulated an increase in fat induced feeding in a genotype-dependent manner using the massduetoadecreaseincorebodytemperatureanduncoupling NOP receptor antagonist SB612111 (Witkin et al., 2014). LY2940094 Inhibits Food Intake in Rodents 501

Mice and rats lacking the NOP receptor have been gener- composition of the diet may have influenced the results ated (Nishi et al., 1997; Köster et al., 1999; Clarke et al., 2001; between studies. Kest et al., 2001; Homberg et al., 2009; Rizzi et al., 2011). Binge eating disorder (BED) affects roughly 1–4% of the Phenotyping of the mouse and rat NOP receptor knockout U.S. population. Currently, atomoxetine, fluoxetine, topiramate, supports a role for endogenous N/OFQ and NOP receptor and baclophen have demonstrated limited efficacy in clinical signaling in the control of numerous CNS functions; however, studies for BED (McElroy et al., 2007a,b; Broft et al., 2007; little data currently exist regarding a feeding or metabolic Grilo et al., 2012). The present work demonstrated that, in phenotype after deletion of the NOP receptor (Higgins et al., preclinical models, the novel NOP receptor antagonist 2002; Depner et al., 2003, Gavioli et al., 2003; Marti et al., LY2940094 effectively inhibited feeding behavior. Several of 2004; Uezu et al., 2004; Chung et al., 2006). NOP receptor these models exhibited a significant hyperphagia, a hallmark knockout mice displayed a reduced preference for sucrose and of BED that is often associated with obesity. These findings a lower intake of high-fat diet under no-choice conditions raise the intriguing possibility that LY2940094 may be (Koizumi et al., 2009). The deletion of the NOP receptor had no effective in the treatment of patients with BED and/or obesity. effect on conditioned place preference or operant responding, Given the existence of a PET ligand for NOP receptors (Pike leading the authors to conclude that reward responses and et al., 2011; Pedregal et al., 2012) and the demonstrated safety motivation for food was unaltered in NOP receptor knockout of LY2940094 in human clinical investigation (Post et al., mice. Moreover, we recently reported that NOP knockout mice 2014), LY2940094 can be used to study the role of NOP exhibit a reduction in fasting-induced feeding (Witkin et al., receptors in eating disorders and metabolic complications. Downloaded from 2014). We found a similar effect of deletion of NOP receptor on fasting-induced feeding in the present study, thus replicating Acknowledgments the earlier findings. Interestingly, the degree of inhibition of The authors thank Steven Kahl and Keyun Chen for assistance. feeding produced by LY2940094 was nearly identical to the This manuscript is dedicated to the memory of our dear friend and reduction in fasting-induced feeding produced by the deletion colleague Conception Pedregal for her never ending enthusiasm and of the NOP receptor itself. Therefore, in agreement with our dedication to the research program. jpet.aspetjournals.org RO data, the doses of LY2940094 that we used likely saturated the NOP receptors in key CNS structures associated with food Authorship Contributions intake. Based on the current findings a more detailed exami- Participated in research design: Statnick, Chen, and Pintar. nation of the metabolic phenotype of NOP receptor knockout Conducted experiments: Ansonoff, Suter, Song, and Hu. mice is warranted. Contributed new reagents or analytical tools: LaFuente, Jimenez, Literature supports that N/OFQ does not appear to mediate Benito, Diaz, Martinez-Grau, Toledo, and Pintar. palatability/reward-based feeding in rodents eating a pre- Performed data analysis: Statnick, Chen, Suter, Song, Hu, and at ASPET Journals on September 23, 2021 Pintar. ferred diet; however, N/OFQ produces hyperphagia in fat- Wrote or contributed to the writing of the manuscript: Statnick, preferring rats (Olszewski et al., 2002). These data led us to Chen, Witkin, Rorick-Kehn, Martinez-Grau, Toledo, and Pintar. choose studying the effect of the nociceptin antagonist LY2940094 on animals eating a highly palatable diet high References in fat and sucrose (HED). 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