P rats were housed individually in TSE LabMaster cages (TSE Systems, Bad Homburg, Germany) with food, water, and 15% ethanol (v/v) available at all times. Water and ethanol intake (in ml) were measured once every 5 min throughout the 12-h dark cycle and recorded for later analysis. In the first experiment, rats (*n* = 10) received vehicle, 3, 10, or 30 mg/kg SB334867 (i.p.), 60 min before onset of the 12-h dark phase of the light-dark cycle, using a within-subjects design. In the second experiment, rats (*n* = 10) received vehicle, naltrexone (10 mg/kg), or LSN2424100 at doses of 10 or 30 mg/kg (i.p.), 60 min before onset of the 12-h dark phase, using a within-subjects design (one rat was excluded from the experiment due to low baseline drinking). In the third experiment, rats (*n* = 32) received vehicle, naltrexone (10 mg/kg), or S-almorexant at doses of 60 or 100 mg/kg (p.o.), 60 min before onset of the dark cycle, using a between-subjects design. Naltrexone was included in the study design as a positive control, since this dose of naltrexone has been shown to effectively reduce ethanol consumption in P rats under these testing conditions. For all experiments, a 60-min pre-treatment period was chosen so that the onset of the dark cycle roughly coincided with the time at which maximal brain concentrations were achieved (data not reported). Consumption of water and ethanol was measured during the first 3 h of the dark cycle, based on the short half-lives and high metabolism of the compounds.

#### *Operant progressive ratio responding in P rats*

P rats were trained and tested 5 days per week in standard rat operant chambers (Med Associates, St. Albans, VT), housed within sound attenuating boxes. Operant chambers measured 30*.*5 × 24*.*1 × 21 cm, with clear Plexiglas front and back walls, modular aluminum sidewalls, a metal bar floor and Plexiglas ceiling. A food cup was located in the center of one sidewall with retractable levers on either side of the food cup. A liquid dipper device allowed the delivery of 0.1 ml of 15% ethanol (v/v) into the food cup. A computer running the MED-IV software package (Med-Associates, St. Albans, VT) controlled stimulus presentations and recorded lever presses. Once subjects were trained to lever press for ethanol reinforcement on a fixed ratio-1 (FR1) schedule of reinforcement, the response requirement for each reinforcement was slowly increased to FR2 and then FR3 over 1–2 weeks. When rats demonstrated a stable level of responding on the FR3 schedule, progressive ratio testing began. The progressive ratio schedule involved increasing response requirements within each session. The response requirement increased from 1 to 2 after three ethanol presentations, and continued to increase by two after every three ethanol presentations (see Rodd et al., 2003). Experimental sessions terminated after 60 min. Total responses on the active and inactive levers, breakpoints [defined as the highest fixed ratio (FR) value reached during the session], and the amount of ethanol consumed (ml; converted to g/kg) were recorded for analysis.

Experiments were conducted using a within-subject design, with 3–4 days washout between administration of different doses, which were counterbalanced using a Latin square design. One group of *n* = 10 rats was used to test the effects of SB334867, LSN2424100, and almorexant on operant responding maintained on a progressive ratio schedule, in separate experiments. Drugs were administered two days per week (Tues and Fri) to allow for washout between subsequent doses. Rats received vehicle, 3, 10, or 30 mg/kg SB334867 (i.p., 30 min prior to the session); vehicle, 3, 10, or 30 mg/kg LSN2424100 (i.p., 30 min prior to the session); or vehicle, 10, 30, or 60 mg/kg almorexant or 60 mg/kg of the inactive enantiomer of almorexant (p.o., 60 min prior to the session). On all other days, rats received progressive ratio operant testing without any drug treatments to maintain operant performance and confirm return to baseline behaviors. One rat was excluded from testing 60 mg/kg almorexant due to observation of a skin rash not related to the study drug.

#### *Binge drinking in C57BL/6J mice*

One week prior to ethanol intake testing, mice were given daily saline injections (i.p.) to acclimate them to handling and injection procedures. Ethanol consumption was assessed using a 4-day drinking-in-the-dark (DID) paradigm during which the water bottle in the home cage was replaced with a single bottle of ethanol (20% v/v) starting 3 h after the onset of the dark cycle. This procedure has been shown to produce high blood ethanol concentrations (BECs) resulting from high levels of ethanol consumption in a relatively short period of time (Rhodes et al., 2005). On the first three days, animals were injected with saline or vehicle 30 min prior to a 2-h period of access to ethanol. On the 4th day, drugs were administered via i.p. injection 30 min prior to the test session, which was extended to 4 h. One cohort of mice was administered vehicle, 3, 10, or 30 mg/kg SB334867 (*n* = 10/dose). A second cohort of mice was tested with vehicle, 15, 30, or 60 mg/kg LSN2424100 (*n* = 9–10/dose). A third cohort of animals was given vehicle, 25, 50, or 100 mg/kg almorexant (*n* = 10/dose). In order to assess resulting BECs, immediately upon removal of ethanol bottles, blood samples were collected from the retro-orbital sinus and centrifuged. The plasma was assayed using an Analox Instruments analyzer (Lunenburg, MA).

In order to assess the specificity of drug effects on ethanol consumption, an additional group of ethanol-naïve animals was tested with sucrose solution (1% w/v) in the same DID paradigm (Days 1–3: 2-h access with saline injections; Day 4: 4-h test session with drug pretreatment). On the 4th day, vehicle, 3, 10, or 30 mg/kg (*n* = 6–7/dose) SB334867 was administered prior to the 4-h access period. During a subsequent week of testing, these same mice were administered either vehicle or 100 mg/kg almorexant (*n* = 14/dose) before the 4-h intake session. In a separate cohort of mice, vehicle or 60 mg/kg LSN2424100 (*n* = 9–10/dose) was administered prior to the 4-h test.

#### *Data analysis*

For subjects treated with SB334867 and LSN2424100 in the home cage drinking studies, ethanol and water intake (g/kg and ml/kg, respectively) during the first 3 h of the dark cycle were calculated and analyzed separately using repeated measures analyses of variance (ANOVAs), with drug dose as a within-subjects factor (IBM SPSS Statistics, Armonk, NY). The positive control, naltrexone, was compared with vehicle using an independent samples *t*-test. For subjects treated with almorexant, ethanol and water intake were analyzed using ANOVAs with drug dose as a betweensubjects factor. The positive control, naltrexone, was compared with vehicle using an independent samples *t*-test. All significant within-subject effects were further explored using paired-samples *t*-tests, except for the between-subjects almorexant study, for which Tukey's HSD test was used.

For subjects treated with SB334867 and LSN2424100 in the operant progressive ratio paradigm, total active lever responses, total ethanol consumption (g/kg), and breakpoints (highest FR value reached) were calculated and analyzed via repeated measures ANOVAs, with dose as a within-subjects factor. The negative control, R-almorexant, was compared with vehicle using a separate paired samples *t*-test. All significant within-subject effects were further explored using paired-samples*t*-tests. Total responding on the inactive levers was negligible, so those data were not analyzed.

For the mouse drinking studies, ethanol consumption during each 4-h test session was expressed in g/kg and subjected to a One-Way ANOVA with dose as a between-subjects factor. BEC data (mg/dl) were analyzed similarly. Sucrose data were expressed as ml/kg and subjected to independent samples *t*tests. Because only the doses of LSN2424100 and almorexant that reduced ethanol consumption were tested with sucrose, the 3 and 10 mg/kg SB334867 data were excluded from the analysis for consistency across compounds.

#### **RESULTS**

#### **HOME CAGE 2-BOTTLE CHOICE DRINKING IN P RATS** *SB334867*

In the first experiment, rats received vehicle, 3, 10, or 30 mg/kg SB334867. A significant main effect of dose was observed [*F(*3*,* 27*)* = 4*.*36, *p <* 0*.*05]. Follow-up paired-samples *t*-tests indicated that the 10 and 30 mg/kg doses of SB334867 significantly reduced ethanol intake relative to vehicle (*p*s *<* 0.05 and 0.01, respectively; **Figure 1A**, filled bars). No significant effects emerged in the analysis of water consumption (*p >* 0*.*05; **Figure 1A**, open bars).

#### *LSN2424100*

In the second experiment, rats received vehicle, 10, or 30 mg/kg LSN2424100, or 10 mg/kg naltrexone. As shown in **Figure 1B**, LSN2424100 did not significantly affect ethanol intake (*p >* 0*.*05; filled bars). However, a paired-samples *t*-test revealed that naltrexone significantly attenuated ethanol intake [*t(*9*)* = 3*.*31, *p <* 0*.*015]. Neither LSN2424100 nor naltrexone produced significant effects on water intake (*p*s *>* 0.05; **Figure 1B**, open bars).

#### *Almorexant*

In the third experiment, rats received vehicle, 60 or 100 mg/kg almorexant, or 10 mg/kg naltrexone in a between-subjects design. Almorexant significantly reduced ethanol intake [*F(*2*,* 20*)* = 3*.*12, *p* = 0*.*05]. A *post-hoc* Tukey's HSD test revealed that only the 100 mg/kg dose of almorexant significantly affected ethanol intake, while effects at the 60 mg/kg dose were not statistically significant (**Figure 1C**, filled bars). An independentsamples *t*-test revealed that naltrexone also significantly reduced ethanol consumption [*t(*13*)* = 3*.*08, *p <* 0*.*01; **Figure 1C**, filled

(*n* = 10) did not significantly influence ethanol or water intake. Naltrexone was included as a positive control at a dose (10 mg/kg) that selectively reduced ethanol intake (indicated by #, *p <* 0*.*05).**(C)**Almorexant (*n* = 8/dose) reduced ethanol intake at the 100 mg/kg dose while also suppressing water intake at the 60 and 100 mg/kg doses. Naltrexone was included as a positive control at a dose (10 mg/kg) that selectively reduced ethanol intake (indicated by #, *p <* 0*.*05). Filled bars indicate ethanol consumption (g/kg) on the left y-axis; open bars indicate water consumption (ml/kg) on the right y-axis. ∗ indicates significant difference (*p <* 0*.*05) relative to vehicle-treated controls.

bars]. Almorexant significantly reduced home cage water intake [*F(*2*,* 21*)* = 10*.*60, *p <* 0*.*01; **Figure 1C**, open bars]. A *post-hoc* Tukey's HSD test revealed that both the 60 and 100 mg/kg doses of almorexant significantly reduced water consumption (*p*s *<* 0.01).

#### **OPERANT PROGRESSIVE RATIO RESPONDING IN P RATS** *SB334867*

Although a trend toward efficacy was observed for SB334867 on progressive ratio responding for ethanol, statistical analysis revealed no significant effects of the drug on the number of active lever responses (data not shown), breakpoints (**Figure 2A**), or total ethanol consumption (**Figure 2B**; all *p*s *>* 0.05).

#### *LSN2424100*

#### The OX2 antagonist LSN2424100 significantly reduced breakpoints [main effect of dose: *F(*3*,* 36*)* = 4*.*61, *p <* 0.01] and resulting ethanol consumption [main effect of dose: *F(*3*,* 36*)* = 6*.*59, *p <* 0*.*01]. *Post-hoc* tests indicated that the 30 mg/kg dose

was significantly different from vehicle (see **Figures 2C,D**). LSN2424100 also significantly reduced active lever presses [*F(*3*,* 27*)* = 3*.*67, *p <* 0*.*05; data not shown].

#### *Almorexant*

Almorexant significantly reduced active lever presses [main effect of dose: *F(*3*,* 24*)* = 25*.*26, *p <* 0*.*001]. Paired-samples *t*-tests indicated that doses of 10, 30, and 60 mg/kg significantly reduced active lever responding (*p*s *<* 0.05; data not shown). A pairedsamples *t*-test indicated that the inactive enantiomer of almorexant did not significantly affect responses on the active lever (*p >* 0*.*05). Similarly, almorexant significantly reduced breakpoints [*F(*3*,* 24*)* = 32*.*32, *p <* 0*.*001], with significant effects at doses of

**FIGURE 2 | Operant progressive ratio responding in P rats.** SB334867 (*n* = 8) did not significantly reduce breakpoints **(A)** or ethanol consumption **(B)** maintained by a progressive ratio operant schedule of reinforcement. The 30 mg/kg dose of LSN2424100 (*n* = 10) reduced the motivation to consume ethanol as indicated by reductions in breakpoints **(C)** and corresponding ethanol consumption **(D)**. **(E,F)** Almorexant (*n* = 10) reduced breakpoints and

ethanol consumption at all doses tested (10, 30, and 60 mg/kg) doses. As expected, the inactive enantiomer (60 mg/kg) did not significantly affect progressive ratio operant responding for ethanol or ethanol consumption. Breakpoint was defined as the highest fixed ratio value reached by rats during the operant session. ∗ indicates significant difference (*p <* 0*.*05) relative to vehicle-treated controls.

10 (*p <* 0*.*01), 30 and 60 mg/kg (*p*s *<* 0.001; **Figure 2E**), while the inactive enantiomer did not (*p >* 0*.*05). Almorexant also significantly attenuated ethanol consumption [*F(*3*,* 24*)* = 32*.*29, *p <* 0*.*001] at doses of 10 (*p <* 0*.*01), 30, and 60 mg/kg (*p*s *<* 0.001; **Figure 2F**). A paired-samples *t*-test indicated that the inactive R-enantiomer of almorexant did not significantly affect ethanol consumption (*p >* 0*.*05).

#### **BINGE DRINKING IN C57BL/6J MICE** *SB334867*

Analysis revealed a main effect of dose [*F(*3*,* 36*)* = 3*.*6, *p <* 0*.*05], with the 30 mg/kg SB334867 dose reducing ethanol intake relative to vehicle-injected controls (**Figure 3A**). BEC data indicated a similar pattern, with a main effect of dose emerging [*F(*3*,* 36*)* = 4*.*4, *p <* 0*.*05]. While *post-hoc* tests revealed no significant differences between vehicle and any doses of SB334867, pairwise comparisons indicated that the 30 mg/kg dose resulted in lower BECs than both the 3 and 10 mg/kg doses (**Figure 3B**). Analysis of sucrose consumption (**Figure 3C**) revealed that 30 mg/kg SB334867 suppressed sucrose intake relative to vehicle [*t(*11*)* = 2*.*74, *p <* 0*.*05].

#### *LSN2424100*

Analysis of ethanol intake on the test day revealed a main effect of dose [*F(*3*,* 35*)* = 4*.*3, *p <* 0*.*05], with the 60 mg/kg dose reducing ethanol consumption relative to vehicle-injected control mice (**Figure 3D**). This reduction was mirrored in BEC data [main effect of dose: *F(*3*,* 35*)* = 6*.*1, *p <* 0*.*01], with the 60 mg/kg dose resulting in lower BECs (**Figure 3E**). Analysis of sucrose consumption data (**Figure 3F**) revealed a significant suppression in mice administered the 60 mg/kg dose relative to vehicle [*t(*17*)* = 8*.*76, *p <* 0*.*001].

#### *Almorexant*

Analysis revealed a main effect of dose [*F(*3*,* 36*)* = 5*.*0, *p <* 0*.*01], with the 100 mg/kg dose reducing ethanol intake relative to vehicle-injected controls (**Figure 3G**). Analysis of BEC data also revealed a main effect of dose [*F(*3*,* 36*)* = 9*.*0, *p <* 0*.*001], with both the 50 and 100 mg/kg doses of almorexant resulting in lower BECs than vehicle-treated control mice (**Figure 3H**). Analysis of sucrose consumption data revealed a trend (*p* = 0*.*085) for almorexant to reduce sucrose consumption, an effect that did not reach statistical significance (**Figure 3I**).

#### **DISCUSSION**

Data from the present series of experiments provide evidence that blockade of OX1 and OX2 receptors reduces ethanol selfadministration in a variety of high-drinking rodent paradigms, although observed effects were dependent on the specific procedures used to evaluate ethanol-seeking behavior. The OX1 receptor antagonist reduced home cage ethanol drinking in rats and binge-like drinking in mice, without influencing progressive ratio operant responding in rats. Blockade of OX2 receptors did not alter home-cage ethanol intake in rats, but did lower breakpoints and reduce ethanol consumption in the progressive ratio procedure in P rats as well as reducing binge-like drinking in mice. Dual antagonism of OX1 and OX2 receptors resulted in reduced ethanol consumption in rats and mice in addition to decreasing breakpoints and ethanol consumption in the operant progressive ratio model. Due to an established role for orexin in the regulation of feeding behavior, it was important to assess the specificity of these drug effects by measuring the ability of the these compounds to alter consumption of another caloric solution. Indeed, results from the present study indicated that some of the test compounds also reduced sucrose consumption in mice.

The majority of previous work exploring the role of orexin in ethanol reward has focused on blockade of OX1 receptors with SB334867, with this compound typically reducing ethanol self-administration (Lawrence et al., 2006; Richards et al., 2008; Moorman and Aston-Jones, 2009; Jupp et al., 2011). In accordance with these findings, we demonstrate here that SB334867 reduced home cage ethanol intake in P rats with a long history of ethanol consumption. SB334867 has been previously shown to reduce breakpoints in an operant progressive ratio procedure in male iP rats (Jupp et al., 2011). In contrast, SB334867 did not significantly alter breakpoints or corresponding ethanol consumption in female P rats in the present study. It is unclear whether this discrepancy can be attributed to sex differences, experimental procedural differences, or differences between respective inbred lines. In the current study, we reported for the first time that SB334867 reduced binge-like ethanol intake in the drinking-inthe-dark procedure in mice. Oddly, the BEC values reported in the vehicle-injected mice were lower than expected given the high ethanol consumption. This apparent disparity may be a consequence of the long length of the testing period. Although not assessed in the current study, different patterns of ethanol consumption over the 4-h period may have resulted in different BEC values. The dose of SB334867 that effectively reduced ethanol intake also suppressed consumption of a 1% sucrose solution. However, previous reports indicate that SB334867 did not affect operant self-administration of a 5% sucrose solution (Richards et al., 2008). It is tempting to speculate that self-administration of the 1% sucrose solution used in the current experiments may be more susceptible to disruption by OX antagonists because it is less palatable to the mice than the 5% sucrose solution in other studies. Further experiments will be required to explore this possibility. Overall, the data reported herein complement and extend previous literature reports demonstrating that SB334867 attenuates ethanol self-administration.

Although previous work has examined OX2 receptor involvement in operant ethanol self-administration and reinstatement procedures, the present study is the first to examine the effects of OX2 antagonism on voluntary home cage ethanol consumption, breakpoints in an operant progressive ratio procedure, and bingelike ethanol drinking using the novel compound LSN2424100. This OX2 antagonist did not alter ethanol consumption under voluntary continuous access conditions in P rats but did reduce breakpoints in the operant progressive ratio procedure, indicating reduced motivation to consume ethanol (Richardson and Roberts, 1996) in alcohol-preferring rats. Consistent with the reduction in operant breakpoints, LSN2424100 also decreased ethanol intake in the progressive ratio model. Our data are consistent with and complement earlier studies in which other selective OX2 receptor antagonists (JNJ-10397049 and

**FIGURE 3 | Binge drinking in C57BL/6J mice. (A)** SB334867 (*n* = 10) reduced ethanol consumption at the 30 mg/kg dose. **(B)** The 30 mg/kg dose of SB334867 resulted in lower BECs relative to both the 10 and 30 mg/kg doses (indicated by #, *p <* 0*.*05). **(C)** In a separate cohort of mice, SB334867 (30 mg/kg) also reduced consumption of a 1% sucrose solution (*n* = 6–7). **(D)** LSN2424100 (*n* = 10) reduced ethanol consumption at the 60 mg/kg dose. **(E)** The same dose resulted in lower BECs relative to vehicle-injected controls.

**(F)** The 60 mg/kg dose of LSN2424100 significantly reduced consumption of a 1% sucrose solution (*n* = 9–10). **(G)** Almorexant (*n* = 10) reduced ethanol intake at the 100 mg/kg dose. **(H)** Both the 50 and 100 mg/kg doses of almorexant resulted in lower BECs. **(I)** In a separate cohort of mice, almorexant (100 mg/kg) did not significantly reduce consumption of a 1% sucrose solution (*n* = 14). ∗ indicates significant difference (*p <* 0*.*05) relative to vehicle-injected controls.

TCS-OX2-29) decreased ethanol self-administration using other operant paradigms in rats (Shoblock et al., 2011; Brown et al., 2013).

Because it is well-known that OX antagonists reduce wakefulness and suppress motor activity in general (e.g., Brisbare-Roch et al., 2007), one might argue that the breakpoint reduction seen in P rats is simply due to reduced locomotor activity. This is unlikely given that the same dose of LSN2424100 (30 mg/kg) did not alter ethanol or water consumption in the home cage drinking procedure reported here. Indeed, although OX receptor antagonists facilitate sleep, they do not produce overt motor impairment or sedative-like effects that are commonly associated with benzodiazepine receptor agonists, such as zolpidem (Steiner et al., 2011). To the contrary, rats receiving OX receptor antagonists, including those tested here, can perform operant and other motor tasks without any observable impairment (Steiner et al., 2011; Rorick-Kehn et al., unpublished observations). That LSN2424100 reduced ethanol self-administration in the operant progressive ratio assay, but not when ethanol was provided under unlimited access conditions, may suggest that OX2 receptor-mediated signaling does not directly modulate ethanol reward. Rather, OX2 receptors may be involved in modulating motivational circuits in the brain that underlie drug-seeking behavior. Indeed, it is not uncommon for drugs to differentially influence ethanol's appetitive/motivational effects vs. consummatory behavior (ethanol drinking *per se*) (e.g., Czachowski et al., 2001, 2002). Alternatively, the differential efficacy of LSN2424100 in these two procedures may reflect the varying ethanol histories of the rats tested in each model.

Inmice, a high dose of LSN2424100 (60 mg/kg) reduced ethanol consumption in the binge-like drinking procedure; however, the same dose also suppressed sucrose consumption, suggesting that this dosewas high enough to produce non-specific effects.Whether the suppression in sucrose intake reflects sleep-promoting effects or a general reduction in consummatory behaviors cannot be determined from the present series of studies. It is interesting that the reduction in sucrose intake (∼85% reduction) was more dramatic than the reduction in ethanol intake (∼36% reduction). The reasons for this difference are not clear. Further work will be necessary to better characterize the nature of these effects.

Antagonism of both OX1 and OX2 receptors by almorexant reduced ethanol drinking in rats with continuous home-cage access and in limited access binge-drinking in mice, and also attenuated breakpoints and ethanol consumption in the progressive ratio model in rats. The data presented here confirm and extend previous reports that almorexant suppressed operant self-administration of both ethanol and sucrose in Long-Evans rats (Srinivasan et al., 2012). Importantly, we demonstrate here that the inactive enantiomer of almorexant did not suppress breakpoints or ethanol self-administration in rats, indicating that the effect was specific to blockade of OX1 and OX2 receptors rather than unknown off-target pharmacological effects. Our data in regard to the specificity of almorexant effects were mixed. Specifically, almorexant reduced home-cage water drinking in rats, suggesting potential non-specific effects on fluid consumption, but it did not significantly attenuate sucrose intake in the mice. Almorexant has previously been shown to reduce operant responding for both ethanol and 5% sucrose when administered systemically; however, when administered directly into the VTA, effects were selective for ethanol (Srinivasan et al., 2012). In the current report, it is unclear whether the reduced water intake in rats, and the tendency for reduced sucrose intake in mice, reflects non-specific consummatory effects or transient sedative effects that may dissipate over the course of the extended drinking session. Additional examination of the selectivity of effects of the compounds tested in the present work, including assays of locomotor activity, is warranted.

The drinking paradigms employed in the present study involved different amounts of total ethanol exposure. Previous studies have reported alterations in the orexin system following long-term ethanol exposure. For example, chronic voluntary ethanol consumption (∼5 g/kg/day for 70 days) has been reported to upregulate hypothalamic preproorexin mRNA in P rats (Lawrence et al., 2006) whereas a reduction in orexin mRNA has been reported after chronic ethanol consumption (∼0.75–2.5 g/kg/day for 28 days) in Sprague-Dawley rats (Morganstern et al., 2010). Several methodological differences between the two studies may account for the seemingly contradictory results, including differences in total daily ethanol exposure (∼5 g/kg/day vs. ∼0.75–2.5 g/kg/day), differences in genetic background (selectively bred P rats vs. Sprague-Dawley rats), and endpoint measured (preproorexin vs. orexin A mRNA). Nonetheless, further studies will be required to determine the impact of longterm ethanol exposure on the brain orexin system, and whether adaptations in orexin signaling resulting from chronic ethanol exposure contribute to the development of addiction. Indeed, others have demonstrated that orexin-A stimulates dopamine cell firing in the VTA, increases dopamine release in the prefrontal cortex (PFC), and potentiates PFC-evoked excitation of VTA dopamine cells (Narita et al., 2006; Vittoz and Berridge, 2006; Moorman and Aston-Jones, 2010). Moreover, Borgland et al. (2006) demonstrated that orexin signaling in the VTA plays a critical role in synaptic plasticity associated with cocaine addiction. The relevance of orexin-mediated signaling in critical processes associated with addiction to drugs of abuse, including ethanol, is beginning to be understood, and will likely be further informed by the development and characterization of additional selective tool compounds from different chemical scaffolds, such as LSN2424100 reported here, that can be used to explore the relative roles of OX1- and OX2-receptor-mediated signaling. Future studies in models of ethanol dependence using selective pharmacological tools may provide valuable information about the therapeutic potential of orexin antagonists for the treatment of alcohol abuse and alcoholism.

#### **ACKNOWLEDGMENTS**

Financial support for P-rat studies was provided by Eli Lilly and Company. Mouse studies were supported by NIH/NIAAA grants to Howard C. Becker (P50 AA010761, U01 AA014095, and U01 AA020929) and the Department of Veterans Affairs. Rachel I. Anderson is supported by NIH/NIAAA grant T32 AA007474.

#### **REFERENCES**

**Conflict of Interest Statement:** Financial support for P-rat studies was provided by Eli Lilly and Company. Benjamin L. Adams, Cynthia D. Jesudason, and Linda M. Rorick-Kehn are employees of, and stockholders in, Eli Lilly and Company.

*Received: 14 November 2013; accepted: 08 February 2014; published online: 25 February 2014.*

*Citation: Anderson RI, Becker HC, Adams BL, Jesudason CD and Rorick-Kehn LM (2014) Orexin-1 and orexin-2 receptor antagonists reduce ethanol self-administration in high-drinking rodent models. Front. Neurosci. 8:33. doi: 10.3389/fnins.2014.00033 This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Anderson, Becker, Adams, Jesudason and Rorick-Kehn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### LSN2424100: a novel, potent orexin-2 receptor antagonist with selectivity over orexin-1 receptors and activity in an animal model predictive of antidepressant-like efficacy

*Thomas E. Fitch\*, Mark J. Benvenga , Cynthia D. Jesudason , Charity Zink , Amy B. Vandergriff , Michelle M. Menezes , Douglas A. Schober and Linda M. Rorick-Kehn*

*Lilly Research Laboratories, Department of Neuroscience, Eli Lilly and Company, Indianapolis, IN, USA*

#### *Edited by:*

*Michel A. Steiner, Actelion Pharmaceuticals Ltd., Switzerland*

*Reviewed by: J. M. Monti, Clinics Hospital, Uruguay Selena Bartlett, Translational Research Institute, Australia Christine E. Gee, University Medical Center Eppendorf-Hamburg,*

*\*Correspondence:*

*Germany*

*Thomas E. Fitch, Lilly Corporate Center DC0510, Eli Lilly and Company, Indianapolis, IN 46285, USA e-mail<EMAIL_ADDRESS>
We describe a novel, potent and selective orexin-2 (OX2)/hypocretin-2 receptor antagonist with *in vivo* activity in an animal model predictive of antidepressant-like efficacy. *N*-biphenyl-2-yl-4-fluoro-*N*-(1H-imidazol-2-ylmethyl) benzenesulfonamide HCl (LSN2424100) binds with high affinity to recombinant human OX2 receptors (*Ki* = 4*.*5 nM), and selectivity over OX1 receptors (*Ki* = 393 nM). LSN2424100 inhibited OXA-stimulated intracellular calcium release in HEK293 cells expressing human and rat OX2 receptors (*Kb* = 0*.*44 and 0.83 nM, respectively) preferentially over cells expressing human and rat OX1 (*Kb* = 90 and 175 nM, respectively). LSN2424100 exhibits good exposure in Sprague–Dawley rats after IP, but not PO, administration of a 30 mg/kg dose (AUC0 6 *h* = 1300 and 269 ng∗ − h/mL, respectively). After IP administration in rats and mice, LSN2424100 produces dose-dependent antidepressant-like activity in the delayed-reinforcement of low-rate (DRL) assay, a model predictive of antidepressant-like efficacy. Efficacy in the DRL model was lost in mice lacking OX2, but not OX1 receptors, confirming OX2-specific activity. Importantly, antidepressant-like efficacy of the tricyclic antidepressant, imipramine, was maintained in both OX1 and OX2 receptor knock-out mice. In conclusion, the novel OX2 receptor antagonist, LSN2424100, is a valuable tool compound that can be used to explore the role of OX2 receptor-mediated signaling in mood disorders.

**Keywords: hypocretin, orexin, neuropeptide, OX2 antagonist, antidepressant, DRL, rat, mouse**

#### **INTRODUCTION**

The orexin (or hypocretin) family of neuropeptides, termed orexin-A (OXA) and orexin-B (OXB), and the receptors to which they bind, were first identified in the late 1990's (De Lecea et al., 1998; Sakurai et al., 1998). The orexin peptides bind to two G-protein coupled receptors, orexin-1 (OX1) and orexin-2 (OX2), with OXA showing roughly equal affinity for OX1 and OX2 receptors and OXB binding preferentially to OX2 receptors (Spinazzi et al., 2006). Although orexin-containing neurons are predominantly localized in the lateral and posterior hypothalamus, they send widespread projections throughout the neuro-axis (Spinazzi et al., 2006). Orexin has been demonstrated to play a role in mediating feeding (Sakurai et al., 1998), regulation of sleep/wake states (Chemelli et al., 1999), addiction and rewardseeking behavior (Richards et al., 2008; Aston-Jones et al., 2009), monoaminergic transmission (Borgland et al., 2008; Ortega et al., 2012), and stress modulation (Boutrel et al., 2005). That many of these functions are often dysregulated in depression has led researchers to propose that orexins may be involved in the pathophysiology of depression (reviewed in Nollet and Leman, 2013).

The exact role of orexins in modulating mood and depressive disorders has proven more difficult to delineate. Early clinical studies evaluating the potential role of orexin in depressive disorders indicated that depressed patients had a blunted diurnal variation in CSF orexin-A levels accompanied by higher orexin-A levels at night, and that these differences were improved with antidepressant treatment, suggesting a link between disrupted orexin signaling and HPA axis disruption associated with depression (Salomon et al., 2003). However, later studies reported contradictory findings, specifically that CSF orexin-A levels were decreased in depressed patients who had attempted suicide (Brundin et al., 2007). To better understand the role of orexin signaling in depression, some researchers turned to putative genetic animal models of depression, although these studies also produced seemingly contradictory findings. For example, it has been demonstrated that Wistar–Kyoto rats, long-regarded as a genetic model of depression based on enhanced sensitivity to stress and reduced sensitivity to selective serotonin reuptake inhibitors (SSRIs; Lopez-Rubalcava and Lucki, 2000), exhibit reduced numbers of orexin neurons, as well as smaller soma size, lower prepro-orexin mRNA expression, and lower OXA levels in various brain areas (Taheri et al., 2001; Allard et al., 2004). The Flinders Sensitive Line (FSL) is another line of selectively bred animals with a prodepressive phenotype characterized by reduced body weight, reduced sexual behavior, disrupted sleep/wake patterns (particularly REM sleep) and exaggerated immobility responses in the Porsolt Forced swim test (Overstreet and Wegener, 2013). In contrast to results in Wistar–Kyoto rats, the FSL rats were found to have a higher number of OX-immunopositive hypothalamic neurons associated with higher immobility in the forced swim test (Mikrouli et al., 2011). Recently, Nollet et al. (2011) reported that unpredictable chronic mild stress was associated with increased aggression and activation of orexin neurons in the dorsomedial hypothalamus, along with concomitant reductions in the number of OX2, but not OX1, receptors in ventral hippocampus, thalamus and hypothalamus. Interestingly, these effects were reversed by chronic treatment with the SSRI antidepressant, fluoxetine (Nollet et al., 2011). The dual orexin receptor antagonist, almorexant, has also been shown to produce antidepressant-like effects in the chronic unpredictable mild stress model (Nollet et al., 2012). While accumulating evidence suggests that orexin neurotransmission may play a role in modulating mood states, little is known about the molecular mechanisms involved.

A more thorough understanding of the involvement of the orexin system in mediating depression, a complex disorder with multiple behavioral sequelae, would benefit from the availability of precise pharmacological probes from multiple chemical scaffolds. These, in turn, could lead to the development of novel pharmacotherapies targeting the orexin system that could prove beneficial in treating depressive disorders. The OX1 antagonist SB334867 (Smart et al., 2001), and the dual orexin antagonist, almorexant (Brisbare-Roch et al., 2007), have been broadly utilized in preclinical experiments. Although OX2-selective antagonists have been reported in the literature, including TCS-OX2-29 (Hirose et al., 2003), JNJ-10397049 (McAtee et al., 2004), and EMPA (Malherbe et al., 2009), very little has been published on these molecules, especially with regard to antidepressant-like efficacy.

Here, we describe a novel OX2 receptor antagonist *N*- ((1*H*-imidazol-2-yl)methyl)-*N*-([1,1 -biphenyl]-2-yl)-4-fluorob enzenesulfonamide hydrochloride (LSN2424100, **Figure 1**), and characterize it in terms of its *in vitro* binding affinity, functional selectivity, and pharmacokinetic properties, and further examine its effects on c-fos expression in the rat prefrontal cortex, a brain region implicated in the pathophysiology of depression (Drevets et al., 2008), in response to restraint stress. We then contrast the effects of this compound in an established animal model predictive of antidepressant-like efficacy, the differential reinforcement of low-rate (DRL) schedule of reinforcement, in both rat (DRL-72; O'Donnell et al., 2005), and mouse (DRL-36; Zhang et al., 2009), as well as mice lacking OX1 and OX2 receptors. The DRL model has been pharmacologically validated for detecting antidepressant-like efficacy using clinical antidepressants across multiple pharmacological classes, including tricyclic antidepressants, SSRIs, norepinephrine reuptake inhibitors, and monoamine oxidase inhibitors as indicated by reduced impulsivity, improved response inhibition, and improved response timing (O'Donnell et al., 2005; Zhang et al., 2009).

#### **MATERIALS AND METHODS DRUGS AND REAGENTS**

*N*-((1*H*-imidazol-2-yl)methyl)-*N*-([1,1 -biphenyl]-2-yl)-4-fluo robenzenesulfonamide hydrochloride (LSN2424100), SB334867, and both (S)-almorexant (ACT-078573; Weller et al., 2005) and its inactive enantiomer (R)-almorexant were synthesized at Lilly Research Laboratories (Indianapolis, IN; see **Figure 1**). LSN2424100 and SB334867 were suspended in a solution of 1% carboxymethylcellulose, 0.25% polysorbate 80, and 0.05% Dow antifoam and injected IP in a volume of 10 ml/kg body weight for mice and 1 ml/kg in rat (LSN2424100 was injected in a 2 ml/kg volume for c-fos studies). (S)-almorexant and (R)-almorexant were suspended in a solution of 1% carboxymethylcellulose, 0.25% polysorbate 80, and 0.05% Dow antifoam and dosed PO in a volume of 2 ml/kg in rat (dosed IP at 10 ml/kg for mice). Imipramine and alprazolam were purchased from Sigma-Aldrich (St. Louis, MO). Imipramine was dissolved in sterile water. Alprazolam was dissolved in 10% 2-hydroxypropyl-βcyclodextrin. Drugs were mixed fresh on the day of the experiment and doses were calculated based on the free base weight.

#### **SUBJECTS**

Adult Male Sprague–Dawley rats weighing 200–225 g (Harlan, Indianapolis, IN for c-fos experiments) were housed 4 per cage with *ad libitum* access to food (Teklad 4% Rat Diet; Harlan Teklad, Madison, WI) and water (except during experimental sessions) and maintained on a 12 h light:dark cycle (lights on at 0600 h). Rats were acclimated to housing conditions for 4 days, followed by sham dosing once daily for 3 days, prior to the experiment. For DRL experiments, male Sprague–Dawley rats weighing between 300 and 350 g at the beginning of the behavioral experiments (Holtzman, Madison, WI) were housed in pairs. For mouse DRL studies, male C57Bl/6 mice (Taconic Farms, Hudson, NY) or mice lacking either OX1 or OX2 receptors, around 8 weeks of age, were obtained from private breeding colonies at Taconic Farms

(Hudson, NY). OX1 and OX2 receptor knockout mice were generated using *in vitro* fertilization of embryos in C57Bl/6 female mice with sperm harvested from male mice obtained from the University of Texas Southwestern Medical Center (Dallas, TX), and backcrossed for at least 10 generations. Mice and rats were housed in separate colony rooms, which were maintained at 22◦C and 60% relative humidity. For rat DRL experiments, water was available for a 20-min period following the daily behavioral session. Based on recommendations from the animal care and use committee, the mouse DRL assay was developed using food deprivation rather than water deprivation. Mice had free access to water except during experimental sessions, were maintained at 85% of free-feeding weight, and received 1 h of free-feeding after each experimental session. All experiments were conducted during the light cycle and in compliance with the Guide for the Care and Use of Laboratory Animals under protocols approved by a local animal care and use committee.

#### **RADIOLIGAND BINDING**

Recombinant human OX1 or OX2 receptors were stably expressed in HEK293 cells and grown in DMEM/F-12 (3:1) supplemented with 5% FBS, 20 mM HEPES, 100 ug/ml Penn/Strep, and 500 ug/ml geneticin. Briefly, the membranes were isolated by homogenizing cell pellet in 30 ml (w/v) 50 mM Tris buffer (pH 7.4) containing Roche Complete EDTA free protease tablets. Membranes were incubated with ∼0.25 nM [125I]-Orexin A (PerkinElmer, Inc., Waltham, MA) for 90 min at 22◦C in polystyrene 96-deep well plates. All binding studies were conducted at a final volume of 200μl. The assay buffer contained 25 mM HEPES, 2.5 mM CaCl2, 1.0 mM MgCl2, 0.5% BSA, and 0.125% BSA (pH 7.4). To generate binding affinity (*Ki*), 11 different compound concentrations were incubated with ∼15μg of OX1 membrane or ∼50μg of OX2 membrane in the presence of [125I]-Orexin A. Compounds were solubilized to make a 10 mM stock in DMSO then diluted to 40μM by placing 4μl into 996μl binding assay buffer. Then 300μl of this solution was placed in column B on a Nunc polypropylene 96-well plate (Thermo Fisher Scientific, Inc., Rochester, NY). Serial dilutions were then made using a Biomek 2000 (Beckman Coulter, Inc., Fullerton, CA) from a starting concentration of 40 μM. Non-specific binding was determined in these experiments using 10μM SB-334867 and LSN2158312 for OX1 and OX2, respectively. All binding was terminated by rapid filtration using a TOMTEC 96-well cell harvester (Hamden, CT) through GF/A filters that had been presoaked with 0.3% polyethyleneimine. The filters were washed with 5.0 ml ice-cold 50 mM Tris buffer (pH 7.4) and air-dried overnight. The dried filters were treated with MeltiLex A (PerkinElmer, Inc., Waltham, MA) melt-on scintillator sheets, and the radioactivity retained on the filters counted using a Wallac 1205 Betaplate (PerkinElmer, Inc., Waltham, MA) scintillation counter. Protein concentrations were measured using Coomassie Protein Plus Assay Reagent (Pierce, Rockford, IL) and serum albumin standards. *Ki*-values from displacement of [125I] orexin-A binding were calculated based on 11-point dilution curves using ActivityBase templates (ID Business Solutions, Ltd., Guildford, Surrey, UK). Reported values are shown as a mean ± the standard error of the mean (s.e.m.).

#### **INTRACELLULAR CALCIUM MOBILIZATION**

Recombinant human OX1 and OX2 receptors were stably expressed in HEK293 cells, or rat recombinant OX1 and OX2 receptors stably expressed in AV12 cells, and assessed for intracellular calcium mobilization using Fluo-3 dye (Molecular Probes, Eugene, OR). Fluo-3 dye was made at 2.2 mM in equal parts of Pluronic f-127 (Molecular Probes, Eugene, OR) and 100% DMSO. It was further diluted to 8 mM in 2.5 mM probenecid loading buffer which was made as described below. The human cell line was grown in DMEM/F-12 (3:1) supplemented with 10% FBS (heat-inactivated), 20 mM HEPES, 1% Penn/Strep, and 4μg/ml Blasticidin (35,000 cells/well). The rat cell line was grown in DMEM supplemented with 10% FBS (heat-inactivated), 20 mM HEPES, 1% Penn/Strep, 4μg/ml Blasticidin, and 1 mM sodium pyruvate (60,000 cells/well). Cells were plated in Biocoat black poly-d-lysine coated clear bottom 96-well plates (Becton Dickinson, Bedford, MA), allowed to attach for 30 min at room temperature, and then grown overnight at 37◦C and 5% CO2 in a humidified incubator. Probenecid loading buffer was made at 250 mM in equal parts of 1 N NaOH and HBSS. It was further diluted to 2.5 mM in HBSS containing calcium and magnesium +20 mM HEPES. The agonist used in this study was Orexin-A (Bachem/Peninsula Laboratories, San Carlos, CA) dissolved in de-ionized water at a stock concentration of 80μM. An EC70 concentration of orexin A in 0.1% BSA (Sigma Aldrich, St. Louis, MO) in HBSS with calcium and magnesium, was used to challenge the antagonists. Compounds were serial diluted 1:3 from a 10 mM stock in DMSO on a Biomek (Beckman Coulter, Inc., Fullerton, CA) and further diluted in HBSS containing calcium and magnesium +0.04% Bacitracin (USB Corp., Cleveland, OH). The assay was performed with the following steps: (1) growth medium was removed and cells were washed with 30μl HBSS (with calcium and magnesium) and then removed. Next, 30μl of dye was added to the wells, and cell plates were incubated in the dark at room temperature for 60 min; dye was removed, cells were washed with 30μl probenecid loading buffer and removed, and then 50μl of probenecid loading buffer was added to the wells. Finally, plates were read on a fluorescence imaging plate reader (FLIPR; Molecular Devices, LLC, Sunnyvale, CA) instrument with a 50μl addition of antagonist; cells were placed in the dark at room temperature for an additional 15 min; then read on FLIPR instrument with a 100μl addition of an EC70 concentration of orexin A (for a total volume of 200μl). Final concentrations of the test compounds were 20μM in 1.25% DMSO and 10μM in 0.625% DMSO for the two different FLIPR reads, respectively. *Kb*-values were calculated based on 10-point dilution curves using ActivityBase templates (ID Business Solutions, Ltd., Guildford, Surrey, UK). Reported values are shown as a mean ± the standard error of the mean (s.e.m.).

#### **RAT EXPOSURE AND UNBOUND FRACTION**

Two male cannulated rats were administered a single 30 mg/kg intraperitoneal (IP) or 30 mg/kg oral (PO) dose of LSN2424100 to determine the pharmacokinetic parameters. Plasma samples were collected at 0.5, 1, 1.5, 2, 4, and 6 h post-dose and analyzed by liquid chromatography coupled to tandem mass spectral detection (LC-MS/MS) to determine the concentrations of LSN2424100. Two cohorts of rats were used in each study, with samples taken from one cohort at 0.5, 1, and 2 h post-dose, and samples taken from the second cohort at 1.5, 4, and 6 h post-dose. The first cohort of animals was sacrificed at 2 h post-dose, and the second at 6 h post-dose to allow for collection of brain samples. The plasma and brain binding of LSN2424100 were determined by equilibrium dialysis at 1μM which were used to calculate unbound concentrations in brain and plasma at 2 h post-dose (Zamek-Gliszczynski et al., 2011).

#### **RESTRAINT-STRESS-INDUCED c-FOS ACTIVATION**

Beginning at 0830 h on the day of the study, groups of rats (*n* = 8 per group) received IP injections of vehicle, 30 mg/kg LSN2424100, or 3 mg/kg alprazolam in a counter-balanced manner and were returned to the home cage. Thirty min later, rats were restrained in a rigid plastic flat-bottom restraint tube (Braintree Scientific, Inc.; Braintree, MA, USA) for 20 min. After the 20 min stress period, rats were moved to a new cage and housed individually. Rats were sacrificed 2 h after stress onset. Separate groups of control animals (*n* = 6 − 8 each) received either vehicle or 30 mg/kg LSN2424100 and were returned to their home cage until sacrifice. Immediately after sacrifice, brains were removed, the medial prefrontal cortex (mPFC), a brain area implicated in the pathophysiology of depression, was dissected from both hemispheres and frozen in 1.5 ml centrifuge tubes in dry ice, and stored at −80◦C for later analysis.

#### **c-Fos ELISA ANALYSIS**

Frozen mPFC tissues were removed from the −80◦C freezer and placed on ice. Tissue was homogenized with a dounce homogenizer. The nuclear extract was prepared from the homogenized samples as per manufacturer's instructions using a CHEMICON ®Nuclear Extraction Kit (Millipore; Billerica, MA, USA). Protein concentrations were determined in the nuclear extracts using the Pierce BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL, USA). Nuclear fractions were then assayed for Fos protein levels using the Pierce c-Fos Transcription Factor Kit (Pierce Biotechnology, Inc., Rockford, IL, USA), according to the manufacturer's instructions. A Wallac 1420 VICTOR luminometer (PerkinElmer; Waltham, MA, USA) was used to measure chemiluminesence in the ELISA plates. Data were analyzed and plotted using GraphPad Prism (GraphPad Software, Inc.; La Jolla, CA, USA).

#### **RAT APPARATUS AND TRAINING**

Sixteen operant-conditioning chambers (30.5 × 24.1 × 29.2 cm; MED Associates, St. Albans, VT) were utilized in conducting the DRL experiments. Each chamber was enclosed in a melamine sound-attenuating cubicle. A white noise generator provided masking noise. The interior of each chamber consisted of three levers mounted on one wall with a house light mounted on the opposite wall. The house light was turned on at initiation of each test session and was turned off at the termination of each session. A water access port was situated next to the lever in the middle of the wall, wherein a reinforced response caused a clicker apparatus to sound paired with a dipper (0.02-ml cup) to be lifted from a water trough to an opening in the floor of the access port for 4 s.

Rats were water deprived for ∼22.5 h before each session. Rats were initially trained under an alternative fixed ratio 1 water reinforcement schedule with a fixed 1-min time schedule for automatic reinforcement. Thus, each response was reinforced, with water also provided every minute in the absence of a response. Rats that did not acquire lever-pressing behavior following three daily 1-h sessions under this schedule were trained using the method of successive approximation. Following acquisition of lever-pressing behavior, rats were trained daily on DRL 18-s sessions for ∼2 weeks, following which they were advanced to DRL 72-s sessions. Responding on these sessions became stable after ∼8 weeks. Experimental test sessions lasted for 1 h and were conducted 5 days/week during light hours.

#### **MOUSE APPARATUS AND TRAINING**

Twelve operant-conditioning chambers (30.5 × 24.1 × 29.2 cm; MED Associates, St. Albans, VT) were used for the DRL experiments. The levers in these chambers were mounted on one wall with an adjacent food magazine next to the lever in the middle of the wall. A reinforced response caused a clicker apparatus to activate and the pellet feeder to deposit one 45-mg sucrose pellet (BioServ, Frenchtown, NJ) into the food magazine. The house light, which was mounted on the ceiling, was turned on when the session began, remained on throughout the entire session, and was turned off at the end of the session. Each experimental chamber was enclosed in a melamine sound-attenuating cubicle and equipped with a white noise generator to provide masking noise.

Each mouse was initially trained under a variable time 60-s operant schedule for 5 days, followed by fixed ratio-1 ratio schedule for 5 days. After the mice had acquired lever-pressing behavior, they were trained during daily DRL 6-s sessions for ∼1 week, then mice progressed through weekly ascending DRL requirements (12, 18, 24, 30 s) before moving to DRL 36-s sessions. The responding on these sessions became stable after ∼6 weeks. Experimental sessions lasted for 45 min and were conducted 5 days/week during light hours.

#### **DELAYED REINFORCEMENT OF LOW-RATE (DRL) DATA ANALYSES**

Drugs were administered to the animals once or twice weekly with at least 1 week between subsequent drugs to minimize possible carryover effects. Drug treatments were administered on Tuesdays and Fridays. No treatments were administered on other test days. Wednesdays were control days. "Control" behavior was calculated as the pooled mean lever presses or reinforcers received during Wednesday sessions across all Wednesdays for the duration of each study. Performance was normalized to percent of control by dividing total responses (or reinforcers) for each drug or vehicle treatment by the mean number of responses (reinforcers) in the respective pooled control condition, multiplied by 100.

The data analyses were conducted on the raw response and reinforcement data. All behavioral data are expressed as the mean ± s.e.m. normalized to the control condition. The effects of LSN2424100 (3–40 mg/kg), SB334867 (3–40 mg/kg), almorexant (10–100 mg/kg) along with its inactive enantiomer (100 mg/kg), and imipramine (1–15 mg/kg) were analyzed using separate One-Way repeated measures ANOVAs on responses and reinforcements, followed by Dunnett's *post-hoc* tests (alpha = 0.05).

**Table 1 |** *In vitro* **binding and functional activity at human recombinant OX1 and OX2 receptors.**

*aRadioligand binding was performed using HEK293 membranes expressing either human OX1 or OX2 receptors in the presence of [*125*I]-orexin A with a 90 min incubation time. Data represent the mean* ± *s.e.m. performed on separate occasions with the number of independent experiments in parenthesis. (R) is the inactive enantiomer of almorexant.*

*bInhibition of calcium mobilization from HEK293 cells stably expressing human or rat OX1 or OX2 receptors, respectively. Functional antagonist activity was determined by measuring the effects of an EC*70 *concentration of orexin-A. Data represent the mean* ± *s.e.m. performed on separate occasions with the number of independent experiments in parenthesis. NT* = *not tested.*

#### **RESULTS**

#### **RADIOLIGAND BINDING AND INTRACELLULAR CALCIUM MOBILIZATION**

Using both an orexin filtration binding assay and a functional intracellular calcium mobilization assay performed in antagonist mode, LSN2424100 demonstrated selectivity for human OX2 receptors stably expressed in HEK293 cells over OX1 receptors (**Table 1**). LSN2424100 was approximately 87-fold more potent at OX2 with a *Ki* of 4.49 nM (OX1 *Ki* = 393 nM) in the receptor binding assay. In the functional assay, LSN2424100 was over 200-fold more potent for human OX2 with a *Kb* = 0*.*44 nM whereas the *Kb* for human OX1 was 90.3 nM. Similar results were obtained in the rat OX receptor cell lines, with rat OX2 and OX1 *Kb* = 0*.*83 and 175 nM, respectively, indicating 210-fold selectivity. Conversely, SB334867 was more selective for the human OX1 receptor (**Table 1**). SB334867 was completely inactive at the OX2 receptor in both the *in vitro* assays. The measured binding affinity of SB334867 for the OX1 receptor was 173 nM. SB334867 showed potent inhibition of intracellular calcium mobilization, with OX1 *Kb* in human and rat = 8.68 and 7.1 nM, respectively. In addition to testing selective orexin agents, we also evaluated the non-selective orexin antagonist, almorexant. The more active S-enantiomer of almorexant demonstrated balanced activity for both human OX1 and OX2 receptors (**Table 1**). The binding affinities of (S)-almorexant for OX1 and OX2 receptors were 21 and 6.9 nM, respectively. Similarly, in the functional assay, (S)-almorexant showed roughly equal potency at inhibiting intracellular calcium mobilization in cells expressing hOX1 and hOX2 receptors, with *Kb*-values of 2.32 and 1.73 nM, respectively. The (R)-enantiomer of almorexant was inactive in both assays.

#### **RAT EXPOSURE AND UNBOUND FRACTION**

Pharmacokinetic data for LSN2424100 in rats are summarized in **Table 2**. LSN2424100 exhibits good exposure in Sprague– Dawley rats after IP, but not PO, administration of a 30 mg/kg dose (AUC0−6 *h* = 1300 and 269 ng∗h/mL, respectively). Mean maximum plasma concentrations observed following a single 30 mg/kg IP and PO dose were 1170 and 196 ng/ml, respectively at 0.5 h post injection. Mean brain concentrations observed following a single 30 mg/kg IP and PO dose were 77.2 and 33.5 ng/g,

#### **Table 2 | Pharmacokinetic datafor LY2424100***a***.**

*an* = *2 cannulated rats/dose.*

*bAbbreviations: AUC*0−6 *h, area under the concentration vs. time curve from time 0–6 h; Cmax , maximum observed drug concentration; tmax , time to maximum observed drug concentration; Fu, fraction unbound in brain/plasma; Cu (unbound or free concentration)* = *Ctotal* × *Fu.*

*cTwo hours post treatment.*

respectively, at 2 h post injection. Unbound fraction in plasma and brain were determined to be 0.0341 and 0.0089, respectively.

#### **c-Fos EXPRESSION**

Administration of LSN2424100 to subjects in home cages produced no effects on c-fos protein levels in the rat prefrontal cortex, compared to vehicle treated subjects (*p >* 0*.*05; **Figure 2**). However, restraint stress invoked a significant increase in c-fos expression in vehicle treated rats [*F(*4*,* 28*)* = 23*.*19; *p <* 0*.*001], and this effect was attenuated by pre-treatment with either LSN2424100 or alprazolam (*p <* 0*.*05; **Figure 2**).

#### **DRL**

In rats, imipramine produced a significant antidepressant-like response, characterized by a decrease in total lever presses emitted (51% of control responding) at 10 mg/kg [*F(*3*,* 68*)* = 10*.*16, *p <* 0*.*0001] with a concomitant increase in reinforcers received [210% of control responding; *F(*3*,* 68*)* = 14*.*47, *p <* 0*.*001; **Figure 3A**]. Doses of 1 and 3 mg/kg had no significant effect on behavior. Raw (non-normalized) data for all DRL experiments are provided in Supplementary Table S1. In mice, imipramine also produced an antidepressant-like signature, with an increase in reinforcers received (330% of control, **Figure 3B**) at 15 mg/kg [*F(*4*,* 60*)* = 7*.*32, *p <* 0*.*001], and a concomitant decrease in lever presses emitted [70% of control; *F(*4*,* 60*)* = 4*.*2, *p <* 0*.*005]. The antidepressant-like effect of imipramine was maintained in mice lacking OX1 receptors, as demonstrated by a dose-dependent increase in reinforcers received up to 15 mg/kg [206% of control; *F(*3*,* 44*)* = 3*.*33, *p* = 0*.*028; **Figure 3C**], and a decrease in lever presses emitted (63% of control) but this difference did not quite reach statistical significance [*F(*3*,* 44*)* = 2*.*64, *p* = 0*.*06; Dunnett's *post-hoc* test, *p* = 0*.*02; **Figure 3C**]. Lower doses of imipramine did not affect lever pressing in mice lacking OX1 receptors. Similarly, imipramine produced antidepressant-like effects in mice lacking OX2 receptors, with a significant effect in both lever presses emitted [*F(*3*,* 44*)* = 6*.*63, *p <* 0*.*001] and reinforcers received [*F(*3*,* 44*)* = 7*.*58, *p <* 0*.*001; 70 and 140% of control, respectively, at 15 mg/kg; **Figure 3D**].

In rats, SB334867 did not produce a significant change in either reinforcers received or total lever presses made, at doses up to 30 mg/kg (**Figure 4A**). Importantly, the positive control used in this test, imipramine, significantly increased reinforcers received [160% of control, *F(*4*,* 24*)* = 9*.*22, *p <* 0*.*001] and decreased lever presses [64% of control, *F(*4*,* 24*)* = 16*.*21, *p <* 0*.*001]. In mice, SB334867 did not significantly affect lever presses emitted or reinforcers received up to 40 mg/kg (*p >* 0*.*05; **Figure 4B**). In mice lacking OX1 receptors, SB334867 produced a significant decrease in lever presses emitted at 20 and 40 mg/kg (75 and 35% of control, respectively; *F(*5*,* 58*)* = 10*.*5, *p <* 0*.*001), while only producing a significant increase in reinforcers received at 20 mg/kg [166% of control; *F(*5*,* 58*)* = 3*.*95, *p <* 0*.*005; **Figure 4C**]. Although an increase reinforcers received was observed at 40 mg/kg (123%), this was not significant. In mice lacking OX2 receptors, SB334867 only produced a disruption of lever pressing at 40 mg/kg [*F(*5*,* 58*)* = 17*.*79, *p <* 0*.*001; **Figure 4D**]. At this dose, lever presses emitted were less than 20% of control, while reinforcers received were 75% of control, indicative of the animals' inability to behave within the scheduled operant parameters.

(S)-Almorexant treatment in rats produced a significant increase in reinforcers received [*F(*3*,* 30*)* = 4*.*09, *p <* 0*.*02; **Figure 5A**], while the reduction in lever presses emitted did not reach statistical significance (*p >* 0*.*05). The inactive enantiomer (R)-almorexant produced no effect on behavior at 100 mg/kg (*p*s *>* 0.05). In mice, (S)-almorexant produced a significant increase in reinforcers received [*>*400% of control; *F(*4*,* 28*)* = 5*.*95, *p* = 0*.*001], while also producing a significant dose-related decrease in lever presses emitted [47% of control; *F(*4*,* 28*)* = 6*.*46, *p <* 0*.*001; **Figure 5B**]. Again, (R)-almorexant produced no significant effects on behavior at 100 mg/kg. In mice lacking OX1 receptors, all doses of (S)-almorexant reduced the number of lever presses emitted [10–60 mg/kg; *F(*4*,* 51*)* = 10*.*16, *p <* 0*.*001] while a significant increase in reinforcers received was observed at 10 and 40 mg/kg [*F(*4*,* 51*)* = 4*.*39, *p <* 0*.*004; **Figure 5C**], although these effects do not appear to be dosedependent. In these animals, (R)-almorexant also produced effects similar to (S)-almorexant, with significant increases in reinforcers received and a concomitant significant decrease in

both LSN2424100 (†††*p <* 0*.*001; *n* = 6) and alprazolam (†*p <* 0*.*05; *n* = 6).

lever pressing [*t(*54*)* = 2*.*04, *p <* 0*.*05 and *t(*54*)* = 2*.*04, *p <* 0*.*05, respectively]. In mice lacking OX2 receptors, the effect of (S) almorexant was completely abolished (*p*s *>* 0.05; **Figure 5D**). In these mice, (R)-almorexant (100 mg/kg) produced a significant decrease in lever presses emitted [65% of control; *t(*54*)* = 2*.*04, *p <* 0*.*05], but did not significantly affect the number of reinforcers received.

When administered to rats, LSN2424100 produced a significant increase in reinforcers received [**Figure 6A**; 170% of control; *F(*4*,* 24*)* = 3*.*13, *p* = 0*.*033] at 30 mg/kg, along with a significant decrease in lever presses made [60 and 70 of control; *F(*4*,* 24*)* = 4*.*64, *p* = 0*.*006] at 10 and 30 mg/kg, respectively. In mice, LSN2424100 produced an increase in reinforcers received (up to 225% of control, **Figure 6B**), but this effect did not reach statistical significance. LSN2424100 treatment did produce a significant decrease in lever presses emitted at 10 mg/kg [50% of control; *F(*3*,* 35*)* = 3*.*28, *p* = 0*.*032]. A higher dose of 20 mg/kg produced a decrease in lever presses emitted, but this result did not reach statistical significance (**Figure 6B**). In mice lacking OX1 receptors, the antidepressant-like efficacy of LSN2424100 was maintained, as demonstrated by a statistically significant increase in reinforcers received at 20 mg/kg [200% of control; *F(*3*,* 36*)* = 2*.*98, *p* = 0*.*044], and a significant decrease in lever presses emitted at this dose [50% of control; **Figure 6C**; *F(*3*,* 36*)* = 4*.*73, *p* = 0*.*007]. In mice lacking OX2 receptors, however, there was no significant change either in reinforcers received or lever presses emitted with LSN2424100 doses administered up to 40 mg/kg (**Figure 6D**), indicating complete loss of antidepressant-like efficacy in OX2 knockout mice.

**FIGURE 3 | Antidepressant-like effect of imipramine on DRL-72-s responding in rats and DRL-36-s responding in mice.** Consistent with literature reports, imipramine produced a significant antidepressant-like effect in both rat DRL-72-s (**A**; *n* = 12) and mouse DRL-36-s models (**B**; *n* = 16), as demonstrated by a significant increase in reinforcers (*p <* 0*.*001 and *p <* 0*.*01, respectively) and significant decrease in total responses emitted (*p <* 0*.*01 and *p <* 0*.*001, respectively). Importantly, the antidepressant-like efficacy of imipramine was not altered in mice lacking OX1 or OX2 receptors (**C,D**, respectively; *n* = 8). ∗*p <* 0*.*05 vs. respective vehicle, Dunnett's post-hoc analyses.

#### **DISCUSSION**

Herein, we describe the pharmacological characterization of a structurally-novel OX2 receptor antagonist, LSN2424100, with high affinity and selectivity for OX2 over OX1 receptors. *In vitro* selectivity data for OX2 over OX1 receptors obtained using radioligand binding and intracellular calcium mobilization assays showed 87- and 205-fold selectivity, respectively (210-fold selectivity in the rat functional assay). Results showed that the two comparator compounds, SB334867 and (S)-almorexant, possessed *in vitro* profiles in the expected ranges, wherein SB334867 showed high affinity and selectivity for OX1 receptors, while the dual orexin antagonist, almorexant, showed roughly equal affinity for OX1 and OX2 receptors. Pharmacokinetic data indicated that LSN2424100 exhibits good exposure in male Sprague–Dawley rats after IP, but not PO, administration of a 30 mg/kg dose (AUC0−6 *h* = 1300 and 269 ng∗h/mL, respectively), suggesting its use as a tool compound to evaluate the role of OX2 receptor signaling mechanisms in preclinical models.

Here, we report that orexin-2 receptor antagonists, including LSN2424100 and almorexant, exhibit antidepressant-like efficacy in the rat DRL-72 sec model, at 20–30 and 100 mg/kg, respectively, as indicated by significant reductions in total lever presses

emitted along with concomitant increases in reinforcers received. Our data are consistent with previous reports that increased OX signaling produced anhedonia-like symptoms in rats (measured by intracranial self-stimulation) (Boutrel et al., 2005), whereas almorexant produced antidepressant-like effects in a model of chronic unpredictable mild stress (Nollet et al., 2012). Interestingly, Nollet et al. (2011) demonstrated that chronic unpredictable mild stress increased activation of dorsomedial hypothalamus/perifornical area (DMH-PFA), where orexin cell bodies are localized. Moreover, they reported that chronic treatment with the SSRI, fluoxetine, reversed the DMH-PFA activation, suggesting a role for orexin signaling in the pathophysiology of depression and antidepressant treatment response (Nollet et al., 2011). The OX1 antagonist SB334867 did not produce antidepressant-like effects in the assays studied here. Together, the pharmacological data suggest that OX2 receptor signaling may be involved in modulating mood states. The results presented here contradict other studies suggesting that increases in orexin-dependent signaling may produce antidepressant-like effects (Ito et al., 2008; Lutter et al., 2008; Scott et al., 2011). The

Dunnett's post-hoc analyses.

obtained (*p <* 0*.*05) by C57Bl/6 mice in DRL-36-s at 100 mg/kg and a significant decrease (*p <* 0*.*05) in total responses emitted at 100 mg/kg, as well as lack of effect of the inactive enantiomer at 100 mg/kg (*n* = 8); **(C)** significant decrease in responses emitted at all doses tested (*p <* 0*.*05) with a concomitant increase in reinforcers received at doses of 10 and 40 mg/kg (*p <* 0*.*05) in mice lacking OX1 receptors (*n* = 8). However, 100 mg/kg of the inactive enantiomer also produced significantly fewer responses (*p <* 0*.*05) with a concomitant increase in reinforcers received (*p <* 0*.*05) in mice lacking OX1 receptors; **(D)** almorexant failed to produce antidepressant-like effects in mice lacking OX2 receptors, up to 100 mg/kg (*n* = 8). The inactive enantiomer (100 mg/kg) significantly reduced total responses emitted in mice lacking OX2 receptors (*p <* 0*.*05), without significantly affecting reinforcers earned (*n* = 8). ∗*p <* 0*.*05 vs. respective vehicle, Dunnett's post-hoc analyses.

reason for this difference is unclear, but may be related to methodological differences between labs. Efficacy in the forced swim test, although useful in many respects, does not always translate to clinical antidepressant efficacy (De Pablo et al., 1989). The DRL model used here has strong construct and predictive validity, and is less likely to produce false positive results (O'Donnell et al., 2005; Zhang et al., 2009). Although less susceptible to false positive results due to motor effects, drugs that dramatically affect food intake, as has been reported for orexin antagonists (Mieda et al., 2006; Boutrel et al., 2010), can affect behavior in the DRL model. However, LSN2424100 was found to have no direct effects on food or water intake (Fitch et al., unpublished results; and see Anderson et al., under review). Alternatively, differences between our study and previous reports may be related to the use of mice lacking the orexin peptide (Lutter et al., 2008) vs. mice lacking individual OX1 or OX2 receptors (current study).

**FIGURE 6 | Antidepressant-like effects of LSN2424100 on DRL-72-s responding in rats and DRL-36-s responding in mice. (A)** dose-dependent increase in the number of reinforcers obtained by male SD rats in DRL-72-s (*n* = 7), with a significant effect at 30 mg/kg (*p <* 0*.*05) and a significant decrease in total responses emitted at 10 and 30 mg/kg (*p <* 0*.*05); **(B)** a significant decrease in total responses emitted by C57Bl/6 mice in DRL-36-s at 10 mg/kg (*p <* 0*.*05; *n* = 8); **(C)** antidepressant-like efficacy of LSN2424100 is maintained in mice lacking OX1 receptors, with a significant increase in reinforcers obtained and a significant decrease in responses at 20 mg/kg (*p <* 0*.*05; *n* = 8); **(D)** antidepressant-like effect of LSN2424100 is lost in mice lacking OX2 receptors, as illustrated by lack of significant effects in either responses emitted or reinforcers received at all doses (*n* = 8). ∗*p <* 0*.*05 vs. respective vehicle, Dunnett's post-hoc analyses.

In addition to demonstrating antidepressant-like efficacy of OX2 antagonists in the rat DRL-72 s model, we extend the findings to demonstrate efficacy in a mouse DRL-36 s schedule of reinforcement. Importantly, we demonstrate similar antidepressant-like efficacy of imipramine in the mouse that closely resembles effects observed in the rat DRL model. In the mouse DRL model, LSN2424100 and almorexant produced antidepressant-like effects, similar to the effects observed in rats (**Figures 5**, **6**). Moreover, the antidepressant-like effects of LSN2424100 and almorexant were completely abolished in mice lacking OX2 receptors, confirming the critical role of OX2 receptor signaling in mediating mood and antidepressant-like efficacy of the orexin antagonists tested here. Importantly, the effects of imipramine were found to be similar in mice lacking either OX1 or OX2 receptors, although efficacy appeared to be blunted in mice lacking OX2 receptors (**Figure 3D**). It is possible that the antidepressant-like phenotype observed in mice lacking OX2 receptors (see non-normalized data in Supplementary Table S1) may be responsible for the apparent blunted efficacy in the normalized data. Nonetheless, the efficacy of imipramine was not lost in mice lacking OX2 receptors, indicating that the antidepressant-like effects of imipramine are not mediated through orexin signaling mechanisms. By extension, our data further suggest that the antidepressant-like efficacy of OX2 and dual orexin receptor antagonists are mechanistically distinct from monoamine-based antidepressants. Considering the sleep disturbances often experienced by depressed individuals, the known side effect of SSRIs to exacerbate or even produce sleep disruption in depressed patients, and the robust sleep-enhancing properties of orexin antagonists (Mayers and Baldwin, 2005; Riemann, 2007; Thase et al., 2010), it seems plausible that selectively inhibiting OX2 receptor signaling may provide additional clinical benefits by simultaneously improving mood and sleep.

The OX1 antagonist, SB334867, appeared void of antidepressant-like activity in rats and mice, although antidepressant-like effects were observed in mice lacking OX1 receptors. Our results differ from previous reports that SB334867 produced antidepressant-like efficacy in the mouse forced swim test and tail suspension test (Scott et al., 2011). It has also been reported that mice lacking OX1 receptors had an antidepressant-like phenotype, an effect which we failed to replicate (Fitch et al., unpublished observations). The reason for the apparent antidepressant-like efficacy of SB334867 in mice lacking OX1 receptors is not known, but could involve either compensatory upregulation of OX2 receptors in mice with constitutive deletion of OX1 receptors, or perhaps some off-target, non-orexin pharmacology of SB334867 that may be either amplified or unmasked in the absence of OX1 receptors. Indeed, off-target pharmacology of SB334867 has already been reported by others, including activity at targets that may produce antidepressant-like effects, such as monoamine transporters, norepinephrine transporter, and 5-HT2C receptors (Gotter et al., 2012). Thus, data from our lab supports previous suggestions that SB334867 should not be considered OX1-selective. At the highest dose of 40 mg/kg, SB334867 produced behavioral disruption in mice lacking OX2 receptors, as indicated by strong reductions in total responding and concomitant reductions in reinforcers received. The mechanism by which SB334867 disrupted behavior in these mice is not known, but may be related to compensatory upregulation of OX1 receptors in these mice, an increase in functional sensitivity of OX1 receptors in the absence of OX2 receptors, or some unidentified off-target pharmacology of SB334867. Further studies will be required to understand these effects in the knockout mice.

Stress is a major trigger of depression and relapse to recurrent depressive episodes (Mazure, 1998). In rats, restraint stress stimulates ventral tegmental area (VTA) dopamine cell activity and c-fos expression in the mPFC, an area with dense reciprocal connections to the VTA that is known to be disrupted in depression (Drevets et al., 2008; Valenti et al., 2011). We demonstrate here that restraint stress-induced c-fos activation in the PFC was significantly attenuated by pre-treatment with LSN2424100. Our data are consistent with previous studies demonstrating that restraint-stress induced mPFC activation, as measured by c-fos expression in both rats and mice (Radley et al., 2006; O'Mahony et al., 2010; Valenti et al., 2011), and extend those results to demonstrate that orexin signaling may be involved in mediating stress responses within corticolimbic circuits. Although we and others have reported c-fos activation in the PFC in response to restraint stress, others have reported that restraint stress did not produce robust c-fos activation of hypothalamic orexin cells (Furlong et al., 2009), suggesting that the orexin system may modulate stress responses in the PFC through indirect pathways rather than by direct connections from hypothalamic cells where orexin is synthesized. While the importance of the PFC in regulating mood is well-established, the potential role of orexin in modulating these circuits is unclear. Further studies will be required to explore the exact mechanism by which orexin signaling may modulate prefrontal cortical activation following restraint stress, which may involve modulation of dopaminergic and noradrenergic neurotransmission (Borgland et al., 2008; Del Cid-Pellitero and Garzon, 2011).

Elucidating the role of the orexin system in modulating the complex physiological and behavioral traits associated with psychiatric disorders, including depression, has been a complex undertaking, confounded by a dearth of selective pharmacological tools. With its suggested involvement in the regulation of vigilance states, feeding and energy homeostasis, drug-seeking behavior, and emotional processing (Mieda et al., 2006; Richards et al., 2008; Aston-Jones et al., 2009; Boutrel et al., 2010; Sinton, 2011; Nollet and Leman, 2013), the orexin system appears to play a critical role in maintaining equilibrium in the mammalian nervous system (Sinton, 2011). In pathological states related to mood, arousal, or consummatory behaviors, the orexin system may present opportunities for pharmacological intervention with potentially beneficial outcomes. Recent research has begun to shed light on the diverse signaling properties of orexins. For example, it is known that OX1 and OX2 receptors can couple, either directly or indirectly, to several G-protein effectors, including Gq, Gi*/*o, and Gs, with the potential to confer diverse signaling properties of either OXA or OXB through various intracellular pathways including phospholipase C, adenylyl cyclase, phospholipase A2, and phospholipase D (reviewed recently by Leonard and Kukkonen, 2014). However, little is known about which molecular pathways mediate the diverse physiological effects of orexins reported in the literature. Research focused on elucidating these mechanisms will benefit from an array of tools providing precise control of component function, including OX2-selective antagonists, which have not been widely tested to date with regard to potential antidepressant-like efficacy. Pharmacological tools, in particular, afford the investigator with an accessible, fast-acting and straightforward methodology for manipulation of receptor functioning to gain insight into the dynamics of receptor-ligand interactions, downstream physiological processes, signaling mechanisms, and behavioral output in preclinical models.

We demonstrate here that LSN2424100 is a novel orexin receptor antagonist with high affinity and selectivity for OX2 receptors and favorable pharmacokinetics following IP doses up to 30 mg/kg, and propose its use as a structurally-unique, synthetically-accessible tool for probing the role of OX2 dependent signaling in rodent models. Moreover, we demonstrate that LSN2424100 blocks stress-induced c-fos activation in the mPFC and produces antidepressant-like efficacy in an established animal model of depression in both rats and mice. Moreover, the antidepressant-like efficacy of LSN2424100 was completely abolished in mice lacking OX2, but not OX1, receptors, indicating a critical role of OX2-receptor-mediated signaling. In conclusion, LSN2424100 represents a functional tool compound for the investigation of OX2-receptor-mediated function, particularly in mood disorders.

#### **SUPPLEMENTARY MATERIAL**

The Supplementary Material for this article can be found online at: http://www*.*frontiersin*.*org/journal/10*.*3389/fnins*.*2014*.* 00005/abstract

#### **ACKNOWLEDGMENTS**

The authors would like to thank Stephen F. Chaney and Lisa A. Foltz for their excellent technical assistance.

#### **REFERENCES**

hippocampus. *J. Neurosci.* 31, 4280–4289. doi: 10.1523/JNEUROSCI.5310-10. 2011

**Conflict of Interest Statement:** All authors are employees of, and stockholders in, Eli Lilly and Company. Financial support for the research conducted in this manuscript was provided by Eli Lilly and Company.

*Received: 16 October 2013; accepted: 09 January 2014; published online: 28 January 2014.*

*Citation: Fitch TE, Benvenga MJ, Jesudason CD, Zink C, Vandergriff AB, Menezes MM, Schober DA and Rorick-Kehn LM (2014) LSN2424100: a novel, potent orexin-2 receptor antagonist with selectivity over orexin-1 receptors and activity in an animal model predictive of antidepressant-like efficacy. Front. Neurosci. 8:5. doi: 10.3389/ fnins.2014.00005*

*This article was submitted to Neuropharmacology, a section of the journal Frontiers in Neuroscience.*

*Copyright © 2014 Fitch, Benvenga, Jesudason, Zink, Vandergriff, Menezes, Schober and Rorick-Kehn. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.*

### The selective orexin receptor 1 antagonist ACT-335827 in a rat model of diet-induced obesity associated with metabolic syndrome

#### *Michel A. Steiner 1 \*, Carla Sciarretta2†, Anne Pasquali 3† and Francois Jenck1*

*1 CNS Pharmacology Neurobiology, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland*

*2 Immunology, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland*

*3 Cardiology, Actelion Pharmaceuticals Ltd., Allschwil, Switzerland*

#### *Edited by:*

*Christopher J. Winrow, Merck, USA*

*Reviewed by: Ciaran J. Faherty, Cadence Pharmaceuticals, USA Arshad M. Khan, University of Texas at El Paso, USA*

#### *\*Correspondence:*

*Michel A. Steiner, Actelion Pharmaceuticals Ltd., Gewerbestrasse 16, 4123 Allschwil, Switzerland e-mail<EMAIL_ADDRESS>
*†These authors have contributed equally to this work.*

The orexin system regulates feeding, nutrient metabolism and energy homeostasis. Acute pharmacological blockade of orexin receptor 1 (OXR-1) in rodents induces satiety and reduces normal and palatable food intake. Genetic OXR-1 deletion in mice improves hyperglycemia under high-fat (HF) diet conditions. Here we investigated the effects of chronic treatment with the novel selective OXR-1 antagonist ACT-335827 in a rat model of diet-induced obesity (DIO) associated with metabolic syndrome (MetS). Rats were fed either standard chow (SC) or a cafeteria (CAF) diet comprised of intermittent human snacks and a constant free choice between a HF/sweet (HF/S) diet and SC for 13 weeks. Thereafter the SC group was treated with vehicle (for 4 weeks) and the CAF group was divided into a vehicle and an ACT-335827 treatment group. Energy and water intake, food preference, and indicators of MetS (abdominal obesity, glucose homeostasis, plasma lipids, and blood pressure) were monitored. Hippocampus-dependent memory, which can be impaired by DIO, was assessed. CAF diet fed rats treated with ACT-335827 consumed less of the HF/S diet and more of the SC, but did not change their snack or total kcal intake compared to vehicle-treated rats. ACT-335827 increased water intake and the high-density lipoprotein associated cholesterol proportion of total circulating cholesterol. ACT-335827 slightly increased body weight gain (4% vs. controls) and feed efficiency in the absence of hyperphagia. These effects were not associated with significant changes in the elevated fasting glucose and triglyceride (TG) plasma levels, glucose intolerance, elevated blood pressure, and adiposity due to CAF diet consumption. Neither CAF diet consumption alone nor ACT-335827 affected memory. In conclusion, the main metabolic characteristics associated with DIO and MetS in rats remained unaffected by chronic ACT-335827 treatment, suggesting that pharmacological OXR-1 blockade has minimal impact in this model.

**Keywords: diet-induced obesity, metabolic syndrome, food intake, orexin, orexin receptor antagonist, ACT-335827, food preference, lipid metabolism**

#### **INTRODUCTION**

Metabolic syndrome (MetS) represents a group of risk factors for diabetes and cardiovascular disease; it is characterized by abdominal obesity plus two of four factors including: elevated circulating triglyceride (TG) levels, reduced circulating high density lipoprotein-associated cholesterol (HDLc) levels, elevated blood pressure, and elevated fasting plasma glucose levels (International Diabetes Federation; http://www*.*idf*.*org). Diet-induced obesity (DIO), which is a major causative factor of MetS, can also result in cognitive impairments and in poorer hippocampus-dependent memory function (Eskelinen et al., 2008; Francis and Stevenson, 2011). The current primary intervention for MetS is to lower body weight through reduced energy consumption, a change of diet composition, and increased physical activity (De Flines and Scheen, 2010). Pharmacological treatments are expected to additionally help patients to restrict over-eating by reducing systemic and brain signals responsible for driving high caloric palatable food intake (Adamo and Tesson, 2008).

Orexin neuropeptides A and B modulate energy balance and metabolic homeostasis (Sakurai, 2006) but also palatable food and sweet reward perception (Di Sebastiano and Coolen, 2012).  OXRs are expressed widely in orexinergic projection areas throughout the brain including the mesolimbic system, implicated in reward/aversion, and including several mid- and hindbrain regions, implicated in regulating energy homeostasis and food intake (Trivedi et al., 1998; Marcus et al., 2001). For instance, some orexin neurons project to the ventral tegmental area (VTA), where they synapse with dopaminergic and GABAergic neurons (Balcita-Pedicino and Sesack, 2007); other orexin neurons innervate neuropeptide Y and pro-opiomelanocortin expressing neurons of the arcuate nucleus (Niimi et al., 2001; Tsuneki et al., 2010). Orexin neurons also receive multiple afferent input (Yoshida et al., 2006) from cortico-limbic regions, involved in either positive or negative emotional processing, as well as from local hypothalamic areas, involved in defense reactions, in autonomic and in circadian regulation (Furudono et al., 2006); in addition, orexin neuron activation is modulated by the anorectic adipokine leptin, by the orectic peptide ghrelin, by circulating non-essential amino acids, and by falling glucose levels (Yamanaka et al., 2003; Williams et al., 2008; Louis et al., 2010; Karnani and Burdakov, 2011). OXR mRNA or protein is also found in the periphery including enteric neurons, stomach, pancreas, adrenal gland, and white and brown adipose tissue (Kirchgessner and Liu, 1999; Blanco et al., 2002; Digby et al., 2006; Adeghate et al., 2010; Skrzypski et al., 2011). The relative functional significance of orexins in controlling energy homeostasis and food intake by acting both in the brain and periphery (Heinonen et al., 2008) has remained elusive but orexin peptide or OXR levels appear to be regulated under a variety of nutritional conditions such as food deprivation, sweetener-induced overconsumption and genetic obesity (Mondal et al., 1999; Karteris et al., 2005; Furudono et al., 2006).

The preferential OXR-1 antagonist SB-334867 has been described to reduce normal food intake in rats during the dark phase (Haynes et al., 2000) by decreasing meal size (Parise et al., 2011) and enhancing satiety onset (Ishii et al., 2005). In addition to impacting quantitative natural feeding the OXR-1 may play a key role in mediating the hedonic component of nutrient intake. SB-334867 centrally injected in the VTA of rats inhibited feeding stimulated by intra-nucleus accumbens injections of a mu-opioid receptor agonist (Zheng et al., 2007). The systemic administration of SB-334867 reduced the overconsumption of a HF diet in satiated rats (Choi et al., 2010), and another OXR-1 antagonist, GSK1059865, reduced the binge-like intake of a HF/S diet (Piccoli et al., 2012). Chronic administration of a high dose of SB-334867 to ob/ob female mice lacking leptin reduced food intake and weight gain (Haynes et al., 2002), and this effect was associated with improvements in glucose homeostasis. Contrarily, OXR-1 deficient mice became similarly obese to wild-type mice when challenged with a HF diet (Funato et al., 2009). However, consistent with the reported effects of chronic SB-334867 treatment, fasting blood glucose and serum insulin levels were improved in obese OXR-1 deficient mice (Funato et al., 2009). Finally, a series of studies exploring SB-334867 in operant responding models also suggest that blocking OXR-1 signaling can reduce the motivation to consume HF/S foods (Borgland et al., 2009; Choi et al., 2010; Cason and Aston-Jones, 2013). Central infusion of the OXR-2 specific agonist [Ala11,D-Leu15]Orexin-B over a period of 14 days decreased consumption of a HF diet and reduced DIO (Funato et al., 2009). Genetically elevating brain levels of orexins also prevented DIO by decreasing food intake and stimulating energy expenditure through a mechanism requiring OXR-2. Therefore, either blocking OXR-1 or activating OXR-2 signaling may provide benefits under conditions of prolonged access to high palatable food that will lead to DIO.

The purpose of our study was to test the effects of the novel OXR-1 antagonist ACT-335827 (Steiner et al., 2013) in a rat model of DIO associated with MetS. In comparison to the frequently used OXR-1 antagonist SB-334867, which may also bind to other neuronal receptors (Gotter et al., 2012; Morairty et al., 2012), ACT-335827 shows greater selectivity among a panel of more than 100 neuronal targets [it only binds to the melatonin MT1 receptor with greater than 50% (i.e., 58%) inhibition at 10µM; (Steiner et al., 2013)]. Affinity of ACT-335827, measured by an intracellular calcium release assay, at the recombinant rat OXR-1 was between 7 and 25 nM (depending on incubation time) and between 630 and 1030 nM at the OXR-2. The calculated free brain concentration in Wistar rats after oral gavage of 300 mg/kg was between 97 nM at 1 h and 166 nM at 6 h following administration. Accordingly, the oral dose of 300 mg/kg was effective in reducing a number of anxiety and stress-related measures in the rat including autonomic activation, schedule-induced polydipsia, and fear-potentiated startle; similar to dual OXR antagonists (Steiner et al., 2012). However, in contrast to dual OXR antagonists, ACT-335827 did not simultaneously induce sleep or hypolocomotion at 300 mg/kg, likely because it did not block OXR-2 at his dose (Steiner et al., 2013).

In the present study MetS was induced in rats exposed to a CAF diet with constant access and free choice between HF/S diet or SC. In addition, highly caloric human snacks were provided 4 times weekly. Once metabolic disturbances were established (13 weeks), rats were treated daily before the onset of the dark phase with 300 mg/kg of ACT-335827 or vehicle for 4 weeks. The effects of chronic ACT-335827 treatment were monitored on energy and water intake, food preference, abdominal obesity, glucose homeostasis, plasma lipid levels, and blood pressure. Hippocampus-dependent cognitive function, which has been shown to be impaired in rodents exposed to DIO (Hwang et al., 2010; Davidson et al., 2012), was also assessed in parallel using a contextual fear-conditioning paradigm.

#### **MATERIALS AND METHODS**

#### **ANIMALS**

Thirty male Wistar rats (Harlan, Horst, NL) weighing 160–180 g (6–7 weeks old) were single-housed upon arrival and acclimated to the Actelion animal facility under a regular 12 h light/dark cycle (lights on at 6:00) for 2 weeks.  All experimental procedures were performed in strict accordance with the relevant licenses issued by the Basel-Land Veterinary office and adhered to Swiss federal regulations on animal experimentation.

#### **DIETS**

After acclimatization, rats were randomly divided into two groups. One group of 10 rats (250–325 g; 8–9 weeks old) continued to receive SC *ad libitum* and served as the control group for the effects of CAF diet feeding. A second group of 20 rats (275–320 g) was given a CAF diet comprised of a free choice between SC and a HF/S diet (Diet 2126: 47% kcal fat, 19.5% kcal protein, 33.5% kcal CHO and 4.6 kcal/g; Provimi Kliba, Kaiseraugst, Switzerland), both provided *ad libitum*. The CAF diet group also received a human snack food 4 days per week of 15 kcal (weeks 1–5 of diet exposure), 30 kcal (weeks 6–13 of diet exposure), or *ad libitum* for 1 h at the beginning of the dark phase (weeks 14 to 18 of diet exposure). Seven types of snacks were provided (see Supplementary Table 1). One snack type was given per day and they were alternated between sweet and savory. The inclusion of human snacks, which are both highly caloric and high in sugar, fat, and salt, can be an important addition to rodent models of DIO, since voluntary over-snacking by humans may potentiate obesity, as well as glycemic and cardiovascular abnormalities (Kimokoti and Brown, 2011; Sampey et al., 2011).

#### **EXPERIMENTAL DESIGN**

Please see **Figure 1** for a schematic outline of the experimental design.

Both groups of animals (SC or CAF fed) were maintained on their respective diets for 13 weeks and their body weights were monitored once weekly. Blood glucose and plasma lipids levels were determined at the end of week 11, after a 16 h fast, and at the beginning of week 13, after a 6–8 h fast, respectively.

Chronic ACT-335827 or vehicle treatment began in week 14 of diet exposure. The CAF-diet fed rats were evenly assigned to a vehicle (CAF-Veh, *n* = 11) or an ACT-335827 (CAF-ACT, *n* = 9) treatment group based on their fasting blood glucose levels obtained in week 11. All SC fed rats received vehicle treatment only (SC-Veh, *n* = 10). ACT-335827 or vehicle was orally administered daily, 2 h before the onset of the dark phase. Body weights were recorded in the afternoon before the start of treatment and once weekly thereafter. Food and water intake in the home cages were continuously measured by an automated system (Phenomaster, TSE systems, Bad Hamburg, Germany).

In week 3 of treatment (week 16 of diet exposure), rats were tested in a contextual fear conditioning (CFC) paradigm. At the beginning of week 4 of treatment (week 17 of diet exposure), blood pressure of the rats was measured in the morning. At the end of the same week, the rats were food deprived (16 h) before an oral glucose tolerance test (oGTT) in the morning. At the beginning of week 18 of diet exposure, the rats were sacrificed by decapitation 24 h after the last ACT-335827 administration and following a mild food deprivation (4–6 h).

#### **DRUGS**

ACT-335827 hydrobromide (Actelion Pharmaceuticals Ltd., Switzerland) was freshly prepared in 10% polyethylene glycol 400/0.5% methylcellulose in water, which served as vehicle (Veh). It was administered orally at 300 mg/kg based on the weight of the free base, in a volume of 5 mL/kg, and administered daily 2 h before the onset of the dark phase.

The 300 mg/kg dose was chosen based on the pharmacokinetics of this compound, which were outlined in the Introduction. At this dose, calculated free brain concentrations (*>*97 nM) were sufficient to allow a significant blockade of OXR-1 (affinity at the rat receptor: 7–25 nM, depending on the assay), without yet blocking the OXR-2 (affinity: 630–1030 nM). This was confirmed by the pharmacological actions of this compound, i.e., reducing stress-related outcomes without inducing sleep at this dose. Following oral administration of ACT-335827 at 300 mg/kg, calculated free brain concentrations were rising from 1 h (97 nM) to 6 h (166 nM), and declined thereafter (1 nM at 24 h). Rats consumed the majority of their food during the active, dark phase, and they increased their eating pattern just before the switch of the light-dark cycle. Thus, the rats were treated 2 h before onset of the dark phase, and also the snacks (the most caloric type of food used in this experiment) were provided at the beginning of the dark phase, in order to assure that the large majority of food intake during this chronic study would take place under full blockade of the OXR-1.

#### **FASTING BLOOD GLUCOSE AND PLASMA LIPID ANALYSIS BEFORE CHRONIC DRUG TREATMENT**

In week 11 of diet exposure, rats were fasted overnight (16 h). Using gentle restraint, a tail blood sample was obtained and

**FIGURE 1 | Schematic overview of the experimental setup.** Rats were maintained on standard chow (SC; *n* = 10) or cafeteria diet (CAF; *n* = 20) for 13 weeks (W). The CAF diet was comprised of a free choice between SC and a high fat/sweet diet, both provided at libitum, and intermittent additional access to human snacks. During these 13 weeks body weight (BW) was measured weekly and blood glucose (BG) and plasma lipid (PL) concentrations were assessed in week 11 and 13, respectively. Chronic vehicle (Veh) or ACT-335827 treatment (ACT; 300 mg/kg, p.o., once daily before onset of the dark phase) started in week 14. All SC fed rats received vehicle treatment only (SC-Veh; *n* = 10). The CAF diet fed rats were divided into a vehicle (CAF-Veh; *n* = 11) and an ACT-335827 (CAF-ACT; *n* = 9) treatment group based on an even distribution of their blood glucose and plasma triglyceride levels, measured in week 11 and 13. During weeks 14–17 the food and water intake and the body weight gain was assessed, and the feed conversion efficiency calculated. In week 16 cognitive hippocampal function was assessed using a contextual fear paradigm. In week 17 blood pressure (BP) measurements and an oral glucose tolerance test (oGTT) were performed. In the beginning of week 18 all rats were sacrificed. Their plasma lipid (PL) and plasma leptin levels were assessed and the weight of white adipose tissue deposits (WATs) and the interscapular brown adipose tissue (iBAT) deposit was measured. See the Methods for further details.

glucose levels were determined using a Glucometer (ACCU-CHEK, Roche Diagnostics, Germany). For plasma lipid analysis in week 13, rats were mildly fasted (6–8 h) and blood samples were obtained by sublingual vein puncture under isoflurane anesthesia, divided between EDTA or heparin containing microtainer tubes, and kept on ice. Plasma was collected by centrifugation (2400 × g for 10 min at 4◦C), placed in pre-chilled tubes and stored at −20◦C. Levels of non-esterified fatty acid (NEFA), total cholesterol, and TG were assayed in plasma from EDTAmixed blood, whereas HDL-cholesterol (HDLc) was quantified in plasma from heparinized blood. All lipids were measured using commercial enzyme assay kits and a fully automated analyzer (Beckman Coulter AU480, Nyon, Switzerland).

#### **CONTEXTUAL FEAR CONDITIONING (CFC)**

A single-training trial CFC paradigm was used to assess the effects of the CAF diet in combination with ACT-335827 treatment on hippocampus-dependent learning addressing simple contextual memory. For the training of conditioned fear, rats were singly placed in lit transparent plastic boxes (27 × 27 × 40 cm) equipped with stainless steel grid shock floors within an enclosed cubicle (Ugo Basile, Comerio, Italy) for 5 min. A 1 mA footshook was then delivered for 2 s. After 2 min the rats were returned to their home cages. 24 h later, rats were re-exposed to the same box for a 10 min testing phase. Freezing behavior (defined as absence of all movements except breathing) was measured using an automated video-tracking system (Anymaze; Stoelting Co., Wood Dale, IL, USA).

#### **BLOOD PRESSURE**

Blood pressure was indirectly measured by blood volume pressure recording (VPR) from the tail using the CODA 8-channel high throughput non-invasive blood pressure system (EMKA Technologies, Paris, France). Animals were pre-warmed at 37◦C. They were then placed in restraining tubes and body temperature was maintained using a heating pad. An occlusion tail-cuff was inserted at the base of the tail and a transducer was placed 1 cm below. Rats underwent 7 cycles of measurements, lasting ∼5 min, before they were returned to their home cage. Following manual reviewing, at least 3 of the 7 measurements derived from clearly defined and smooth VPRs were selected for further analysis. Based on this criteria, two CAF diet fed rats treated with ACT-335827, and one CAF diet fed rat treated with vehicle, were excluded from analyses. The systolic and diastolic blood pressures were derived by taking the mean of the 3 selected values. The mean blood pressure was calculated using the following formula: (systolic pressure - diastolic pressure)/3 + diastolic pressure.

#### **ORAL GLUCOSE TOLERANCE TEST (oGTT)**

An oGTT was performed between 8:00 and 12:00 at the end of the fourth week of treatment (week 17 of diet exposure) on overnight fasted rats (16 h). In the morning, tail vein blood was collected from rats of all treatment groups in random order for determination of fasting glucose levels (as described above). Approximately 30 min later, a freshly prepared solution of glucose (2 g/kg/5 mL in water) was orally administered to the rats in a staggered manner, and blood was again collected for glucose quantification 30, 60, 120, and 180 min following oral glucose administration.

#### **TISSUE AND BLOOD COLLECTION AT SACRIFICE**

Eighteen to Twenty hours after the last ACT-335827 or vehicle administration, rats were mildly fasted for 4–6 h before sacrifice by decapitation. Trunk blood was collected, divided between EDTA- and heparin- containing tubes, and kept on ice for ∼2 h before centrifugation. Plasma was collected into pre-chilled tubes and stored at −20◦C for lipid quantification (as described above) or at −80◦C for leptin analysis by a commercially available leptin detection assay (Mesoscale, Gaithersburg, USA). Epididymal, abdominopelvic, and mesenteric white adipose tissues (WATs), as well as intrascapular brown adipose tissue (iBAT) were then rapidly isolated and weighed.

#### **STATISTICAL ANALYSIS**

For each rat, food intake in grams was converted to energy intake in kcal. For the time course analysis of energy and water intake and diet preference, kcal values or water volumes (mL) were cumulated over either 48 h or weekly. Body weight gain was expressed as the percentage change from baseline weight at the start of diet exposure or from the beginning of chronic treatment. The area under the curve (AUC) of the time course of blood glucose changes for each rat during the oGTT was calculated using the trapezoid rule. The weekly feed efficiency of each rat was determined by normalizing body weight gain to the total kcal intake of a given week.

For all measures, mean values and standard errors of the mean (s.e.m.) were calculated per treatment/diet group. Before the beginning of chronic treatment, the blood glucose and plasma TG levels of the CAF diet groups were compared to each other and to those of the SC fed group using unpaired two-tailed *t*-tests, except for diet preference and weight gain, which was analyzed using a Two-Way analysis of variance (ANOVA). Following the start of chronic vehicle or ACT-335827 treatment, for all readouts the effects of CAF diet feeding *per se* were determined by comparing CAF diet fed vehicle treated rats with SC fed vehicle treated rats using unpaired *t*-tests or Two-Way ANOVAs. The effects of ACT-335827 were also determined for all readouts by comparing vehicle-treated and ACT-335827-treated CAF fed rats using unpaired *t*-tests, and Two-Way or Three-Way ANOVAs where appropriate. CAF fed ACT-335827-treated rats were not compared to SC-fed vehicle-treated rats. *Post-hoc* mean comparisons were made using Tukey or Bonferroni tests. All statistical analyses were performed using GraphPad Prism 5.03 or Statistica (version 9) software, and statistical significance was accepted at *p <* 0*.*05.

#### **RESULTS**

#### **PROFILE OF DIET GROUPS BEFORE TREATMENT ONSET**

Over the 13 weeks of diet exposure, the body weight gain of CAF diet fed rats was significantly greater than that of SC fed rats [*Diet*: *F(*1*,* 28*)* = 12*.*20, *p <* 0*.*05; *Time* × *Diet*: *F(*12*,* 336*)* = 3*.*42, *p <* 0*.*05; **Figure 2A**]. *Post-hoc* analyses revealed significant group differences in weeks 8, 12, and 13 of diet exposure (**Figure 2A**).

Fasting glucose levels of CAF diet fed rats were significantly elevated at week 11 when compared to SC fed rats [*t(*30*)* = 2*.*3, *p <* 0*.*05]. This parameter was used to divide CAF diet fed rats in two subgroups with a similar mean and distribution of glucose

standard chow; *CAF*, cafeteria diet; *HF/S*, high fat/sweet; *Veh*, vehicle; *ACT*, ACT-335827).

levels (**Figure 2B**) that were then assigned to receive either vehicle or ACT-335827 upon the start of treatment in week 14. Both subgroups showed similar significantly elevated fasting glucose levels compared to SC fed rats [CAF(Veh) vs. SC: *t(*19*)* = 2*.*14, *p <* 0*.*05; CAF(ACT) vs. SC: *t(*17*)* = 2*.*39, *p <* 0*.*05; CAF(Veh) vs. CAF(ACT): *t(*18*)* = 0*.*11, *p* = 0*.*91].

At week 13 of diet exposure plasma TG levels were also found elevated in both groups fed a CAF diet as compared to SC [CAF(Veh) vs. SC: *t(*19*)* = 3*.*18, *p <* 0*.*01; CAF(ACT) vs. SC: *t(*17*)* = 2*.*18, *p <* 0*.*05] (**Figure 2C**). TG levels did not differ between the CAF diet fed groups [*t(*18*)* = 0*.*57, *p* = 0*.*56]. Plasma levels of NEFA, total cholesterol, HDLc, or the proportion of HDLc in total cholesterol were not elevated in CAF diet fed rats compared to SC diet fed rats (data not shown).

Finally, before the start of chronic drug treatment in week 14, a 48 h assessment of the total energy intake of the HF/S diet and SC on days when no snacks were presented (**Figure 2D**) was performed. The two groups of rats exposed to the CAF diet and the rats fed SC did not differ in their total kcal intake. With respect to food choice, both of the CAF diet fed groups consumed significantly more of the HF/S diet (77.3% of total calories) than SC (22.7% of total calories) [*Food*: *F(*1*,* 18*)* = 53*.*11, *p <* 0*.*05], but they did not differ in their level of preference [*CAF diet* treatment *Group* × *Food*: *F(*1*,* 18*)* = 1*.*97, *p* = 0*.*18]. With respect to water intake, the three groups of rats did not significantly differ (Supplementary Figure 1A).

In summary, rats fed the CAF diet gained more weight and showed elevated fasting blood glucose and plasma TG levels compared to rats fed SC. Forty-eight hours before the onset of chronic ACT-335827 or vehicle administration, the two CAF diet fed groups did not differ from each other in their total kcal and water intake and in their preference of the HF/S diet over SC.

#### **ACT-335827 REDUCES THE PREFERENCE FOR A HF/S DIET WITHOUT AFFECTING WEEKLY TOTAL ENERGY INTAKE, AND INCREASES BODY WEIGHT GAIN AND FEED CONVERSION EFFICIENCY**

In general, all three experimental groups slightly reduced their total energy intake over the 4 weeks of vehicle or ACT-335827 treatment [SC-Veh vs. CAF-Veh: *Time*: *F(*3*,* 57*)* = 17*.*2, *p <* 0*.*05; CAF-Veh vs. CAF-ACT: *Time*: *F(*3*,* 34*)* = 21*.*57, *p <* 0*.*05; **Figure 3A**]. Vehicle treated rats fed a CAF diet did not differ in their weekly total kcal intake compared to vehicle treated rats fed SC [*Diet*: *F(*1*,* 19*)* = 3*.*13, *p* = 0*.*09; *Diet* × *Time*: *F(*3*,* 57*)* = 0*.*26, *p* = 0*.*85]. Chronic administration of ACT-335827 did not affect the total kcal intake of the CAF diet compared to vehicle treatment [*Treatment*: *F(*1*,* 18*)* = 0*.*001, *p* = 0*.*98; *Treatment* × *Time*: *F(*3*,* 54*)* = 0*.*92, *p* = 0*.*44]. Both vehicle and ACT-335827 treated rats fed the CAF diet consumed significantly more of the HF/S diet than SC or snack [*Food*: *F(*2*,* 36*)* = 157*.*4, *p <* 0*.*05]. Analysis of the contribution of each food type to the total kcal intake averaged over 4 weeks of vehicle treatment revealed that the rats obtained in average 74% of their total kcal from the HF/S diet, 18% from SC, and 8% from snacks. Rats chronically treated with ACT-335827 reduced their preference for the HF/S diet over SC [*Treatment* × *Food*: *F(*2*,* 36*)* = 8*.*22, *p <* 0*.*05], irrespective of week of treatment [*Treatment* × *Food* × *Time*: *F(*6*,* 108*)* = 0*.*13, *p* = 1*.*28].

More detailed analyses of the weekly energy intake of each food type separately over the 4 weeks of treatment revealed that ACT-335827 significantly increased the intake of SC as compared to vehicle [*Treatment*: *F(*1*,* 18*)* = 11*.*24, *p <* 0*.*05], and this effect became stronger over time [*Treatment* × *Time*: *F(*3*,* 54*)* = 3*.*08, *p <* 0*.*05; **Figure 3B**]. Indeed, *post-hoc* comparisons showed that ACT-335827 treatment significantly increased SC intake in weeks 3 and 4. Conversely, ACT-335827 treatment significantly decreased the intake of the HF/S diet as compared to vehicle [*Treatment*: *F(*1*,* 18*)* = 7*.*47, *p <* 0*.*05], irrespective of week of treatment [*Treatment* × *Time*: *F(*3*,* 54*)* = 0*.*742, *p* = 0*.*53; **Figure 3C**]. Finally, both the vehicle and ACT-335827 treated rats consumed a similar amount of the snack food over the 4 weeks of treatment [*Treatment*: *F(*3*,* 54*)* = 1*.*38, *p* = 0*.*26; *Time*: *F(*3*,* 54*)* = 2*.*66, *p* = 0*.*06; *Treatment* × *Time*: *F(*3*,* 54*)* = 1*.*38, *p* = 0*.*25; **Figure 3D**].

Vehicle treated CAF diet fed rats and SC fed rats did not differ in their weight gain over the 4 weeks of treatment [*Time*: *F(*4*,* 19*)* = 14*.*91, *p <* 0*.*05; *Diet* × *Time*: *F(*1*,* 76*)* = 0*.*48, *p* = 0*.*74;

**Figure 3E**]. In the CAF diet fed groups, chronic ACT-335827 treatment significantly increased weight gain over 4 weeks compared to vehicle treatment [*Treatment*: *F(*1*,* 18*)* = 6*.*29, *p <* 0*.*05; *Treatment* × *Time*: *F(*4*,* 72*)* = 5*.*42, *p <* 0*.*05]. *Post-hoc* analysis revealed that this increase was due to significantly greater weight gains by the ACT-335827 treated rats in weeks 3 and 4.

indicated time points by *post-hoc* test following ANOVA. **(E)** Body weight

Normalization of weekly energy intake to absolute body weight revealed that CAF diet fed rats treated with vehicle were not hyperphagic compared to SC fed rats or to ACT-335827 treated rats over the 4 weeks of treatment (data not shown). However, examination of the efficiency of feed conversion into body weight *CAF*, cafeteria diet; *Veh*, vehicle; *ACT*, ACT-335827; *HF*/*S*, high fat/sweet). gain of vehicle treated CAF diet fed and SC fed rats revealed a significant reduction for both groups over 4 weeks of treatment from a positive efficiency to a negative efficiency [*Time*: *F(*3*,* 19*)* = 21*.*9, *p <* 0*.*05], but no effect of diet and no diet × time interaction [*Diet*: *F(*1*,* 19*)* = 0*.*07, *p* = 0*.*80; *Diet* × *Time*: *F(*3*,* 57*)* = 1*.*88, *p* = 0*.*14; **Figure 3F**]. Similarly, both groups of CAF fed rats showed decreasing feed efficiency over the 4 weeks of treat-

ment [*Time*: *F(*3*,* 19*)* = 22*.*4, *p <* 0*.*05]. However, ACT-335827 treatment inhibited this decrease compared to vehicle treatment [*Treatment*: *F(*1*,* 19*)* = 5*.*00, *p <* 0*.*05], which was more pronounced during the first 3 weeks [*Treatment* × *Time*: *F(*3*,* 54*)* = 2*.*65, *p* = 0*.*06] and reached significance in week 3 (*p <* 0*.*05; *post-hoc* test).

In summary, rats consuming a CAF diet and chronically treated with ACT-335827 slightly decreased their preference for a HF/S diet over SC without changing their weekly total kcal intake and snack consumption. CAF diet fed rats treated with ACT-335827 gained significantly more weight over 4 weeks compared to vehicle treatment, likely due to increased feed conversion efficiency.

#### **ACT-335827 INCREASES WATER INTAKE IN CAF DIET FED RATS**

Similar to the total food intake, overall water intake slightly decreased over the 4 weeks of treatment with vehicle or ACT-335827 independent of diet [SC-Veh vs. CAF-Veh: *Time*: *F(*3*,* 57*)* = 18*.*91, *p <* 0*.*05; CAF-Veh vs. CAF-ACT: *Time*: *F(*3*,* 54*)* = 11*.*3, *p <* 0*.*05; Supplementary Figure 1B]. Furthermore, vehicle treated rats fed CAF diet drank significantly less water than vehicle treated rats fed only SC, independent of week of treatment [*Diet*: *F(*1*,* 19*)* = 10*.*95, *p <* 0*.*05; *Diet* × *Time*: *F(*3*,* 57*)* = 0*.*03, *p* = 0*.*93]. In CAF fed rats, ACT-335827 treatment significantly increased water intake compared to vehicle treatment, largely irrespective of the week of administration [*Treatment*: *F(*1*,* 18*)* = 12*.*39, *p <* 0*.*05; *Treatment* × *Time*: *F(*3*,* 54*)* = 2*.*38, *p* = 0*.*08]. The effect reached statistical significance in the second and third week of treatment (*p <* 0*.*05; *post-hoc* test).

#### **NO DETECTABLE EMOTIONAL OR COGNITIVE IMPAIRMENTS DUE TO CAF DIET EXPOSURE ALONE OR IN COMBINATION WITH CHRONIC ACT-335827 TREATMENT**

Consumption of energy rich diets high in fat and/or sugar has been shown to impair learning and memory, particularly through their effects on hippocampal function. A CFC task was performed after 3 weeks of chronic ACT-335827 or vehicle treatment (17 weeks following the onset of CAF diet exposure).

During the fear conditioning training the rats of all groups responded with a similar amount of freezing over the 2 min following the presentation of a 1 mA foot shock (data not shown). When returned to the aversive conditioned context 24 h later, vehicle treated CAF-fed rats displayed a high level of freezing (∼70%) over the entire 10 min of testing that was similar to that of both the vehicle treated rats fed SC [*t(*19*)* = 0*.*37, *p* = 0*.*72] and the ACT335827 treated rats fed a CAF diet [*t(*18*)* = 0*.*61, *p* = 0*.*55; **Figure 4**].

#### **CAF DIET-INDUCED GLUCOSE INTOLERANCE IS NOT AFFECTED BY ACT-335827 TREATMENT**

The effect of CAF diet exposure alone and in combination with ACT-335827 treatment on glucose homeostasis and tolerance were assessed. Following a 16 h fast, the blood glucose levels of vehicle-treated CAF fed rats were slightly elevated from those of SC fed vehicle treated rats, but the difference was not significant [*t(*18*)* = 0*.*77, *p* = 0*.*45; **Figure 5A**]. In addition, ACT-335827 treatment of rats fed a CAF diet did not alter fasting glucose levels compared to vehicle treatment [*t(*18*)* = 1*.*91, *p* = 0*.*07].

Upon oral glucose challenge, blood glucose levels were significantly elevated in vehicle treated CAF fed rats compared to

**FIGURE 4 | Effect of CAF diet and ACT-335827 on cognition in week 16.** Percentage of time spent freezing over a 10 min test of contextual conditioned fear. Mean ± s.e.m. (*n* = 9–11 per group). No statistical difference between groups was revealed. (*SC*, standard chow; *CAF*, cafeteria diet; *Veh*, vehicle; *ACT*, ACT-335827).

**FIGURE 5 | Glucose homeostasis in week 17. (A)** Oral glucose tolerance test (oGTT) performed on overnight fasted SC and CAF fed rats treated with Veh or ACT. Whole blood glucose was measured before (0 min) and at the indicated time points after the oral (po) administration of 2 mg/kg glucose (arrow). ∗*p <* 0*.*05 vs. CAF-Veh at the indicated time point by *post-hoc* test following ANOVA. Mean ± s.e.m. (*n* = 8–11) per group. Due to an incomplete blood sample set, data from one SC-Veh and one CAF-Veh rat had to be omitted because of repeated measures within subjects analyses. **(B)** AUC calculated individually for each rat. ∗*p <* 0*.*05 by *t*-test. The CAF-ACT group was statistically not different from the CAF-Veh group. (*SC*, standard chow; *CAF*, cafeteria diet; *Veh*, vehicle; *ACT*, ACT-335827; *po*, per os; *AUC*, area under the curve; *ns*, not significant).

SC fed rats [*Diet*: *F(*2*,* 25*)* = 9*.*85, *p <* 0*.*05; **Figure 5A**], but the shape of the time course up to 180 min after challenge was not affected [*Diet* × *Time*: *F(*8*,* 100*)* = 1*.*33, *p* = 0*.*24]. *Post-hoc* analyses revealed that the effect was most pronounced at the glucose peak (60 min after challenge), which was significantly higher in CAF fed rats than in SC fed rats. Analysis of the AUCglucose confirmed the glucose intolerance of the CAF-fed rats compared to SC-fed rats [*t(*18*)* = 2*.*72, *p <* 0*.*05]. Chronic ACT-335827 treatment in combination with CAF diet feeding showed a trend to impair glucose tolerance but did not significantly alter the levels or time course of blood glucose elevations upon challenge [*Treatment*: *F(*1*,* 17*)* = 4*.*28, *p* = 0*.*054; *Treatment* × *Time*: *F(*4*,* 68*)* = 0*.*23, *p* = 0*.*92; **Figure 5B**], or the AUCglucose [*t(*17*)* = 1*.*80, *p* = 0*.*09].

#### **CAF DIET-INDUCED ELEVATION IN BLOOD PRESSURE IS NOT ALTERED BY CHRONIC ACT-335827 TREATMENT**

Hypertension is correlated with central obesity and is an aspect of MetS. Compared to vehicle treated rats fed a SC diet, vehicle treated rats consuming a CAF diet for 17 weeks had significantly elevated diastolic [*t(*18*)* = 2*.*16, *p <* 0*.*05], systolic [*t(*18*)* = 2*.*53, *p <* 0*.*05], and mean blood pressure [*t(*18*)* = 2*.*37, *p <* 0*.*05; **Figure 6**]. Chronic ACT-335827 treatment did not affect the elevation in blood pressure due to CAF diet feeding.

 The main visceral fat deposits in male rodents are the epididymal depot, the adominopelvic depot, and the mesenteric depot.  Moreover, the proportion of body weight represented by the combined mass of all three visceral fat deposits was significantly higher in vehicle treated rats fed a CAF diet compared to those fed SC [*t(*19*)* = 2*.*36, *p <* 0*.*05; **Figure 7D**]. In addition to WAT, the CAF diet fed rats treated with vehicle also had significantly larger deposits of iBAT compared to vehicle treated SC fed rats [*t(*19*)* = 3*.*0, *p <* 0*.*05; **Figure 7E**]. Chronic treatment with ACT-335827 did not significantly alter the elevations in visceral WAT depots and iBAT mass resulting from CAF diet feeding as compared to vehicle treatment.

#### **ACT-335827 INCREASES THE PROPORTION OF PLASMA HDLc LEVELS, BUT DOES NOT ALTER ELEVATED PLASMA TG LEVELS DUE TO A CAF DIET**

Plasma markers of MetS include reduced levels of HDLc and elevated TG levels. At the time of sacrifice following 4 weeks of vehicle or ACT-335827 treatment (and 18 weeks of diet exposure), plasma levels of HDLc (**Figure 8A**), total cholesterol

**FIGURE 6 | Blood pressure in week 17.** Diastolic, systolic, and mean blood pressures of SC or CAF diet fed rats treated with Veh or ACT. Horizontal bars represent the mean ± s.e.m. (*n* = 7–10) per group. Reliable data was not attainable from 1 CAF-Veh rat and from 2 CAF-ACT rats, and were therefore omitted from analyses. ∗*p <* 0*.*05 by *t*-test. No statistical difference was revealed between the CAF-ACT and the CAF-Veh group. (*SC*, standard chow; *CAF*, cafeteria diet; *Veh*, vehicle; *ACT*, ACT-335827).

(**Figure 8B**), or the proportion of HDLc relative to total cholesterol (**Figure 8C**) were not different between vehicle treated rats fed CAF diet or SC. However, plasma TG levels were significantly higher in vehicle treated CAF diet fed rats compared to SC fed rats [*t(*18*)* = 2*.*70, *p <* 0*.*05; **Figure 8D**]. As expected from elevations in WAT and iBAT mass due to CAF diet feeding reported above, plasma levels of leptin were significantly elevated in these rats compared to SC fed rats [*t(*18*)* = 2*.*72, *p <* 0*.*05; **Figure 8E**]. Finally, plasma levels of NEFA did not differ significantly between vehicle treated rats fed SC or CAF (**Figure 8F**).

ACT-335827 treatment of CAF diet fed rats did not significantly affect plasma levels of TG [*t(*18*)* = 0*.*61, *p* = 0*.*55], cholesterol, NEFA, Plasma HDLc or leptin compared to vehicle treatment. However, the proportion of HDLc in the total plasma cholesterol was found significantly elevated [*t(*18*)* = 3*.*84, *p <* 0*.*05; **Figure 8C**].

#### **DISCUSSION**

#### **CAF DIET FEEDING AS A MODEL OF MetS**

In humans, over-consumption of palatable food rich in fat, sugar, and salt facilitates MetS development. Under a CAF diet comprised of a choice between a HF/S diet and SC and an additional snack 4 times weekly, rats developed major signs of human MetS, including abdominal adiposity, elevated TG and fasting blood glucose levels, glucose intolerance, and elevated blood pressure when compared to rats fed only SC. Conversely, plasma levels of NEFA, total cholesterol, HDLc, and the ratio between the latter two, were not altered. These findings are in agreement with those of other DIO studies in rodents reporting a similar induction of partial abnormalities associated with MetS in man [e.g., (Sampey et al., 2011)]. An impairment of hippocampus-dependent memory function induced by DIO, which might be a consequence of altered blood-brain barrier integrity (Davidson et al., 2012) and/or a reduction of growth factors and proteins responsible for synaptic plasticity (Woo et al., 2013), has been demonstrated both in human and rodents (Kanoski and Davidson, 2011). Still, hippocampus memory function, which was assessed in the present study by employing a CFC paradigm, was not altered in rats fed a CAF diet compared to rats fed SC. Using a one-trial conditioning protocol and a shock intensity similar to ours, Hwang et al. showed that male mice fed a normal diet responded with ∼50% freezing during the 6 min test of conditioned fear, whereas this was reduced to ∼20% in mice fed a HF diet for 1 year (Hwang et al., 2010). A CAF diet exposure of only 4 months as in our study may have been insufficient to cause learning impairments in this test.

#### **EFFECT OF CHRONIC ACT-335827 TREATMENT IN THE MetS MODEL**

ACT-335827 treatment began in week 14 of CAF diet exposure, a time point when rats displayed elevated fasting blood glucose and plasma TG levels. Our results indicate that 4 weeks of ACT-335827 treatment did not significantly alter metabolic abnormalities and adiposity generated by CAF diet exposure alone, despite the animals showing an improved diet composition due to a slight but significant reduction in preference for the HF/S diet over SC. This reduction in HF/S diet intake upon OXR-1 blockade is

**FIGURE 7 | Fat mass at sacrifice.** The effect of SC or CAF diet exposure on the mass of epididymal **(A)**, abdominopelvic **(B)**, and mesenteric **(C)** visceral fat deposits in rats treated with Veh or ACT. **(D)** Normalization of cumulated visceral fat mass (in **A–C**) to body weight (BW) per rat and expressed as a

percentage. **(E)** Mass of intrascapular brown adipose tissue (iBAT). Mean ± s.e.m. (*n* = 9–11 per group). ∗*p <* 0*.*05 by *t*-test. No statistical difference was revealed between the CAF-ACT and the CAF-Veh group. (*SC*, standard chow; *CAF*, cafeteria diet; *Veh*, vehicle; *ACT*, ACT-335827; *ns*, not significant).

consistent with other studies in rats suggesting a role for OXR-1 activation in positively regulating the motivation to consume palatable foods (Zheng et al., 2007; Choi et al., 2010). A decrease in reward perception under ACT-335827 treatment might also be expected to decrease intake of highly palatable snacks. However, consumption of the human snacks (presented for 1 h, 4 times weekly), was not affected by the drug. ACT-335827 administration may thus, be ineffective at reducing the binge-like intake of highly palatable foods under conditions, where either another type of palatable food (the HF/S diet) is freely available, or where no additional intermittent periods of stress or food-restriction occur. Using the OXR-1 antagonist GSK1059865, Piccoli et al. showed that OXR-1 signaling is only involved in the binge-like intake of highly palatable food if an additional specific stressor component (e.g., smelling but no access to the palatable food) is present (Piccoli et al., 2012).

The reduced overall food intake among all experimental groups during weeks 3 and 4 of vehicle or ACT-335827 treatment may be due to the performance of the CFC test to assess cognition (in week 3), and a blood pressure analysis and an overnight period of fasting before the oGTT (in week 4). These procedures were likely a source of stress on the rats, which may have affected their energy consumption and led to a slight decline of body weight gain in week 4. It is unlikely that those procedures confounded the overall conclusions of the current study with regard to OXR-1 blockade because all experimental groups (SC-Veh, CAF-Veh, and CAF-ACT) were simultaneously affected. Moreover, the overall pattern of the different diet consumption, which was displayed by the different groups already in week 2 of chronic treatment (until when no additional potentially stressful experimental procedures had been performed) was actually maintained in weeks 3 and 4 as well (see **Figure 3A**). Whether the additional exposure to environmental stressors during weeks 3 and 4 actually intensified the preference of the CAF-ACT treated rats for consuming more of the SC as compared to the CAF diet, cannot be excluded.

Despite affecting HF/S food preference vs. SC, ACT-335827 had no impact on total calorie intake because the decrease in HF/S food intake was entirely compensated for by an increase in SC intake. As a result, given that chronic ACT-335827 treatment also increased body weight gain, feed efficiency was enhanced. Although not directly tested, these results suggest that ACT-335827 administration may reduce energy expenditure. Funato et al. (2009) showed that energy expenditure is reduced in OXR-1 deficient mice under low fat, but not high fat, feeding conditions. Conversely, ob/ob female mice showed even increased energy expenditure under chronic treatment with SB-334867 administered at 30 mg/kg (Haynes et al., 2002). The reason for these discrepancies remains unclear, but biological compensation, phenotypically expressed in knockout animals, and sex or species differences are likely explanations that can be considered. Moreover, the OXR-1 preferential antagonist SB-334867 has recently been tested by two independent groups for selectivity (Gotter et al., 2012; Morairty et al., 2012): these studies suggest that at high doses as applied by Haynes et al. (2002), SB-334867 could additionally interact with a number of other brain targets.

Orexins also stimulate BAT thermogenesis, but the individual role played by the OXR-1 in modulating BAT function is unclear (Haynes et al., 2002; Sellayah et al., 2011; Tupone et al., 2011). BAT is important for plasma TG clearance, which improves insulin resistance due to DIO (Bartelt et al., 2011). In the current study the elevated weight gain and feed efficiency seen with ACT-335827 treatment under CAF diet feeding was not associated with an altered elevation in iBAT mass or a change in plasma TG levels compared to vehicle treatment. This might suggest that BAT was functioning normally in ACT-335827 treated rats challenged with a CAF diet. However, UCP-1 levels, which serves as a marker of BAT thermogenesis (Haynes et al., 2002; Sellayah et al., 2011), were not analyzed to confirm this conclusion. An alternative possibility for the reduced energy expenditure is that ACT-335827 had an inhibitory effect on non-exercise activity thermogenesis, which can be generated by orexin A (Novak et al., 2006; Novak and Levine, 2009).

Interestingly, the elevated body weight gain and increased feed efficiency seen over 4 weeks of ACT-335827 treatment were not associated with an increase in visceral fat mass compared to vehicle treatment. Thus, the weight gained by ACT-335827 treated-rats must have occurred in fat deposits not analyzed (e.g., subcutaneous) and/or in non-fat tissues. Blood pressure of CAF diet fed rats also remained unchanged by treatment.