A Novel and Selective Nociceptin Receptor (NOP) Agonist (1-(1-((cis)-4-isopropylcyclohexyl)piperidin-4-yl)-1H-indol-2-yl)methanol (AT-312) Decreases Acquisition of Ethanol-Induced Conditioned Place Preference in Mice

Abstract

Background: Nociceptin/orphanin FQ, the endogenous peptide agonist for the opioid receptor-like receptor (also known as NOP or the nociceptin receptor), has been shown to block the acquisition and expression of ethanol (EtOH)-induced conditioned place preference (CPP). Here, we report the characterization of a novel small-molecule NOP ligand AT-312 (1-(1-((cis)-4-isopropylcyclohexyl)piperidin-4-yl)-1H-indol-2-yl)methanol) in receptor binding and GTPγS functional assays in vitro. We then investigated the effect of AT-312 on the rewarding action of EtOH in mice using the CPP paradigm. Further, using mice lacking the NOP receptor and their wild-type controls, we also examined the involvement of NOP in the effect of AT-312. Motivational effects of AT-312 alone were also assessed in the CPP paradigm.

Methods: Female mice lacking NOP and/or their wild-type controls received conditioning in the presence or absence of the NOP agonist [AT-312 (1, 3, and 10 mg/kg) or the control NOP agonist SCH221510 (10 mg/kg)] followed by saline/EtOH for 3 consecutive days (twice daily) and tested for CPP in a drug-free state on the next day.

Results: Our in vitro data showed that AT-312 is a high-affinity, selective NOP full agonist with 17-fold selectivity over the mu opioid receptor and >200-fold selectivity over the kappa opioid receptor. The results of our in vivo studies showed that AT-312 reduced EtOH CPP at the lowest dose (1 mg/kg) tested but completely abolished EtOH CPP at higher doses (3 or 10 mg/kg) compared to their vehicle-treated control group. AT-312 (3 mg/kg) did not alter EtOH-induced CPP in mice lacking NOP, confirming that AT-312 reduced EtOH CPP through its action at the NOP receptor. AT-312 (3 mg/kg) did not induce reward or aversion when administered alone, showing that the novel small-molecule NOP agonist shows efficacy in blocking EtOH-induced CPP via the NOP receptor.

Conclusions: Together, these data suggest that small-molecule NOP agonists have the potential to reduce alcohol reward and may be promising as medications to treat alcohol addiction.

Keywords: AT-312; Alcohol Reward; Ethanol-Induced Conditioned Place Preference; NOP Agonist; NOP Knockout Mouse.

Figure 1. Structures of NOP agonists AT-312 and SCH221510

Figure 2. The effect of AT-312, a novel NOP agonist, on ethanol-induced CPP in C57BL/6J mice

Data are mean (±S.E.M.) of the amount of time that mice spent in the drug-paired chamber (DPCh) before (D1) (preconditioning test day) and after (D5) conditioning (post-conditioning test day). Mice treated with vehicle or AT-312 (1, 3 or 10 mg/kg, s.c.) 15 min before ethanol on the conditioning days. *P<0.05, indicates a significant increase in the amount of time in the DPCh on D5 vs. D1; ++P<0.01, +++P<0.001, significantly different from the control group on D5.

Figure 3. The action of AT-312, a novel NOP agonist, on ethanol-induced CPP in mice lacking NOP and their wild-type littermates/controls

Data are mean (±S.E.M.) of the amount of time that mice spent in the drug-paired chamber (DPCh) before (D1) (preconditioning test day) and after (D5) conditioning (post-conditioning test day). Mice lacking NOP [NOP (-/-)] and their wild-type littermates [NOP (+/+)] were treated with vehicle (left panel) or AT-312 (10 mg/kg, right panel) 15 min before ethanol on conditioning days. ***P<0.001; *P<0.05 DPCh vs. VPCh

Figure 4. Motivational effect of AT-312, a novel NOP agonist, in the place conditioning paradigm

Data are mean (±S.E.M.) of the amount of time that mice spent in the drug-paired chamber (DPCh) on test days before (D1) and after (D5) conditioning. Mice received vehicle or AT-312 (3 mg/kg, right panel) 15 min before saline on the conditioning days.

Figure 5. Effects of SCH221510 (SCH; 10 mg/kg) on ethanol CPP in C57BL/6J mice

Data are mean (±S.E.M.) of the amount of time that mice spent in the drug-paired chamber (DPCh) on test days before (D1) and after (D5) conditioning. Mice were treated with vehicle or SCH221510 15 min before ethanol on the conditioning days. *P<0.05, significant difference in the amount of time between D5 vs D1 for the vehicle-treated group

Figure 6. Effects of pentobarbital (Pento; 25 mg/kg) on ethanol CPP in C57BL/6J mice

Data are mean (±S.E.M.) of the amount of time that mice spent in the drug-paired (DPCh) and vehicle-paired chamber (VPCh) on test days before (D1) and after (D5) conditioning. Mice were treated with vehicle or pentobarbital 15 min before ethanol on the conditioning days. *P<0.05, significant difference in the amount of time that mice spent in the DPCh vs. VPCh on D5.