A selective orexin-1 receptor antagonist attenuates stress-induced hyperarousal without hypnotic effects

Abstract

Orexins (OXs) are peptides produced by perifornical (PeF) and lateral hypothalamic neurons that exert a prominent role in arousal-related processes, including stress. A critical role for the orexin-1 receptor (OX1R) in complex emotional behavior is emerging, such as overactivation of the OX1R pathway being associated with panic or anxiety states. Here we characterize a brain-penetrant, selective, and high-affinity OX1R antagonist, compound 56 [N-({3-[(3-ethoxy-6-methylpyridin-2-yl)carbonyl]-3-azabicyclo[4.1.0]hept-4-yl}methyl)-5-(trifluoromethyl)pyrimidin-2-amine]. Ex vivo receptor binding studies demonstrated that, after subcutaneous administration, compound 56 crossed the blood-brain barrier and occupied OX1Rs in the rat brain at lower doses than standard OX1R antagonists GSK-1059865 [5-bromo-N-({1-[(3-fluoro-2-methoxyphenyl)carbonyl]-5-methylpiperidin-2-yl}methyl)pyridin-2-amine], SB-334867 [1-(2-methyl-1,3-benzoxazol-6-yl)-3-(1,5-naphthyridin-4-yl)urea], and SB-408124 [1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]urea]. Although compound 56 did not alter spontaneous sleep in rats and in wild-type mice, its administration in orexin-2 receptor knockout mice selectively promoted rapid eye movement sleep, demonstrating target engagement and specific OX1R blockade. In a rat model of psychological stress induced by cage exchange, the OX1R antagonist prevented the prolongation of sleep onset without affecting sleep duration. In a rat model of panic vulnerability (involving disinhibition of the PeF OX region) to threatening internal state changes (i.e., intravenous sodium lactate infusion), compound 56 attenuated sodium lactate-induced panic-like behaviors and cardiovascular responses without altering baseline locomotor or autonomic activity. In conclusion, OX1R antagonism represents a novel therapeutic strategy for the treatment of various psychiatric disorders associated with stress or hyperarousal states.

Fig. 1.

Chemical structure of OX1R antagonists compound 56, GSK-1059865, SB-408124, and SB-334867.

Fig. 2.

Characterization of [³H]compound 56. (A and B) Saturation study: total, specific, and nonspecific binding of [³H]compound 56 to CHO or HEK-293 cells stably expressing the human (A) or rat (B) OX1R receptor with increasing radioligand concentration. Nonspecific binding was defined as that remaining in the presence of 10 μM almorexant. Vertical lines show the S.E.M. Data are the mean of three (rat OX1R) or four (human OX1R) experiments. (C) Digitized computer images of the distribution of [³H]compound 56 binding sites in coronal rat brain sections at three different levels; nonspecific binding was determined in the presence of 10 μM GSK-1059865. Color represents relative levels of optical density ranging red > yellow > green > blue > black. Scale bar, 0.25 cm. Amg, amygdala; Cx, cortex; Hip, hippocampus; Hyp, hypothalamus; LC, locus coeruleus TT, tenia tecta.

Fig. 3.

Ex vivo brain OX1R binding autoradiography by compound 56, GSK-1059865, SB-334867, and SB-408124. (A) Duration of occupancy for OX1R antagonists postsubcutaneous administration in rats (10 mg/kg). (B) Occupancy versus dose measured at 0.5 hour. Results are expressed as average percentage receptor occupancy versus vehicle-treated rats ± S.E.M. (n = 3). OX1R occupancy was measured in the tenia tecta.

Fig. 4.

Effects of the OX1R antagonist compound 56 on sleep parameters in rats. NREM latency (A), NREM duration (B), REM latency (C), and REM duration (D) are determined for the 6-hour period after oral dosing (10 mg/kg) at the onset of the dark phase. Results are expressed in minutes and are represented as means ± S.E.M. of eight animals.

Fig. 5.

REM sleep-promoting effects of the OX1R antagonist compound 56 in OX2R KO. REM sleep latency and duration in OX2R KO (A), OX2R WT (B), and NREM sleep latency and duration in OX2R KO (C) and OX2R WT (D) for the 6-hour period after oral dosing (30 mg/kg) during the light phase are expressed in minutes. Values are means ± S.E.M. of seven OX2R KO and five OX2R WT mice. *P < 0.05 and **P < 0.01 versus vehicle as determined by paired Student’s t test for each genotype.

Fig. 6.

Effects of the OX1R antagonist compound 56 on cage-exchange stress-induced hyperarousal in rats and ACTH release in mice. Vehicle or compound 56 was administered subcutaneously (10 mg/kg in rats and 30 mg/kg in mice) 30 minutes prior to cage exchange or control condition. NREM latency (A), REM latency (B), and total sleep (C) in rats for the first 2 hours are expressed in minutes and are represented as means ± S.E.M. of seven animals (four treatment conditions, crossover design). (D) ACTH release in mice is expressed in picograms per milliliter of serum and is represented as the mean ± S.E.M. of 11–13 animals per condition (four treatment conditions). Statistical significance (*P < 0.05, **P < 0.01, and ***P < 0.00) was based on one-way ANOVA followed by Newman-Keuls multiple comparison test.

Fig. 7.

The effects of systemic subcutaneous injections of 3-,10-, and 30-mg/kg doses of compound 56 on NaLac-induced panic-associated autonomic responses [MAP (A), HR (B), and CBT (D)] and behavioral responses [social interaction duration (C) and general motor activity (E)] in rats made panic-prone with chronic reductions of GABA synthesis in the PeF region (i.e., local l-AG infusions). Line graphs are expressed as average percentage ± S.E.M. (n = 9/group). ⁺Between-subjects differences using a Fisher’s least significant difference post-hoc test that was protected with an ANOVA at each time point and significance with a one-way ANOVA with repeated measures was determined as P < 0.05. *Within-subjects differences using a Dunnett’s post-hoc test against baseline with a repeated measures P < 0.05. Bar graph in (C) is expressed as the average percentage ± S.E.M. (n = 9, 9, 8, 9, and 9); + and * indicate between-subjects differences using a Fisher’s least significant difference post-hoc test that was protected with an ANOVA P < 0.05. There was no significant baseline HR, MAP, CBT, or general motor activity between groups (see legends on figures). (F) Coronal brain section from a standard stereotaxic atlas of the rat brain (Paxinos and Watson, 2005) illustrating cannula placements (black circles) in the PeF region and medial nuclei. BPM, beats per minute; bsln, baseline; DA, dorsal hypothalamic area; DMN, dorsomedial hypothalamic nucleus; f, fornix; LH, lateral hypothalamus; mt, mammillothalamic tracts; veh, vehicle.