Small molecule orexin agonist ROXA-47 enhances learning and memory in mice

Abstract

Aging affects multiple physiological functions including physical activity behavior, the sleep-wake cycle, and cognition, processes which are regulated in part by lateral hypothalamic orexin neurons. Loss of orexin peptides and neurons is associated with aging, as well as conditions such as type-1 narcolepsy, which are characterized by altered cognitive function. Direct brain administration of orexin-A improves cognitive function; however, the cognitive effects of orexin agonists delivered systemically have not been studied, in part due to the lack of small molecule agonists suitable for systemic administration. In this study, we developed a metabolically stable small molecule dual agonist ROXA-47 with low nanomolar potencies at both OX1R and OX2R. The effects on cognition of ROXA-47 and a previously reported OX2R agonist YNT-185 were measured by peripheral injections in 6 mo. and 12-mo. old mice using two-way active avoidance (TWAA) and/or the contextual object recognition task (CORT). To determine the individual receptor effect of ROXA-47 on TWAA and CORT memory, OX1R (SB-334867) or OX2R (JNJ-10397049) antagonists (5 mg/kg, i.p.) were injected prior to ROXA-47 (40 mg/kg, i. p.). ROXA-47 and YNT-185 decreased latency and increased total responses in the TWAA, while both ROXA-47 and YNT-185 enhanced discrimination index in the CORT, indicating improved cognition in 6 mo. old and 12-mo. old mice. ROXA-47-enhanced TWAA and CORT memory was attenuated by SB334867 but not JNJ-10397049, suggesting that ROXA-47 improves cognitive function in mice primarily via OX1R activation. These results suggest that small molecule orexin agonists may have therapeutic potential for age-related cognitive decline.

1. Introduction

The lateral hypothalamus (LH) orexin (OX) neurons are implicated in sleep/wake regulation and play critical roles in cognitive functions including learning and memory (Mavanji et al., 2017; Stanojlovic et al., 2019a; Stanojlovic et al., 2019c). The orexin neurons produce orexin-A and B (also known as hypocretins 1 and 2) neuropeptides, which are the endogenous ligands for two G protein-coupled receptors (GPCRs), orexin 1 (OX1R) and orexin 2 (OX2R) (de Lecea et al., 1998; Sakurai et al., 1998). Orexin-A is a 33 amino acid peptide with two disulfide bridges and binds both OX receptors with equal affinity, whereas orexin-B is a 28 AA linear peptide with ~10-fold selectivity for OX2R (Sakurai et al., 1998; Lee et al., 1999). Orexin-expressing neurons are limited in number and located predominantly in a small area of the lateral hypothalamus (Peyron et al., 1998; Sakurai et al., 1998; Date et al., 1999; Nambu et al., 1999). The nerve fibers of orexin neurons project throughout the central nervous system (CNS) (Peyron et al., 1998; Sutcliffe and de Lecea, 2002; de Lecea and Sutcliffe, 2005; Sakurai, 2005), including multiple midbrain and forebrain areas crucial for cognitive processes such as prefrontal cortex (PFC), and hippocampus (HC) (Peyron et al., 1998), and the orexin neurons interact with other neural systems essential to cognitive functions such as the basal forebrain cholinergic system (Fadel and Frederick-Duus, 2008; Boschen et al., 2009; Arrigoni et al., 2010; Fadel and Burk, 2010).

Orexin deficiency causes narcolepsy with cataplexy (Nishino et al., 2001; Ripley et al., 2001), a condition known to produce excessive daytime sleepiness as well as disrupted cognition. Reduced orexin levels and signaling are also found in conditions including aging, early Alzheimer's and Parkinson's diseases, which are characterized by cognitive impairment and decline (Naumann et al., 2006; Fronczek et al., 2007; Thannickal et al., 2007; Fronczek et al., 2012; Oh et al., 2019). A direct association has been observed between cerebrospinal fluid (CSF) orexin-A levels and cognitive function in humans (Shimizu et al., 2020). Similarly, orexin knockout (KO) mice were found to exhibit impaired spatial working memory (Dang et al., 2018). The O/A3 transgenic mice, in which OX neurons degenerate by 3 months of age, display significant deficits in long-term social memory (Yang et al., 2013).

Consistently, central administration of orexin-A enhances learning and memory (Aitta-Aho et al., 2016; Mavanji et al., 2017; Burk et al., 2019; Stanojlovic et al., 2019a; Calva and Fadel, 2020; Um and Lim, 2020). Treatment with orexin-A improved short-term spatial memory in mice (Aitta-Aho et al., 2016), and attenuated learning and memory impairment in capsaicin-treated rats (Kooshki et al., 2017). Similarly, orexin-A (i.c.v.) effectively improved memory retention in young and old SAMP8 mice, which have age-related deficits in learning and memory and an increased level of beta amyloid expression (Jaeger et al., 2002). We have also shown that orexin-A injection into HC or chemogenetic activation of orexin neurons reverses memory impairments in orexin loss models (Mavanji et al., 2017; Stanojlovic et al., 2019a; Stanojlovic et al., 2019c). Lastly, nasal administration of orexin-A, which bypasses the blood-brain barrier (BBB), restored social memory in orexin/ataxin (O/A3) transgenic mice, a model of orexin neuron loss (Yang et al., 2013; Weinhold et al., 2014; Calva and Fadel, 2020). Collectively, these results support orexin agonism as a promising intervention for orexin deficiency-related cognitive dysfunction.

Due to the inherent limitations of peptides [particularly for CNS targets such as the orexin receptors (Takahashi et al., 1999; Fujiki et al., 2003)], including limited BBB penetration, unfavorable metabolic stability, and susceptibility to protease cleavage, there is mounting interest in small molecule orexin receptor agonists (Turku et al., 2016). In 2015, YNT-185 was reported as an OX2R selective agonist (Nagahara et al., 2015; Nagase et al., 2016), which, when i.p. or i.c.v. administered, promoted wakefulness in WT mice and suppressed cataplexy-like episodes in orexin KO and orexin neuron-ablated mice (Irukayama-Tomobe et al., 2017). Another OX2R agonist, TAK-925, demonstrated clinical proof of concept for improving excessive daytime sleepiness through OX2R agonism and informed the development of subsequent oral OX2R agonists. Consistent with this wake-promoting mechanism, recent clinical developments report that oral OX2R agonists improve objective wakefulness and daytime sleepiness in narcolepsy populations, including oveporexton (TAK-861), which met all primary and secondary endpoints in two Phase 3 narcolepsy-1 (NT1) trials (FirstLight; NCT06470828 and RadiantLight; NCT06505031) (Dauvilliers et al., 2025). Similarly, alixorexton (formerly ALKS 2680), also has shown positive topline Phase 2 efficacy in NT1/narcolepsy-2 (NT2) trials (Vibrance-1; NCT06358950 and Vibrance-2; NCT06555783) (Shahzadi et al., 2025). Taken together, these findings support an orexin-driven therapy for age related cognitive decline. We recently discovered the first dual agonist RTOXA-43, which activates both OX1R and OX2R with equal potencies (OX1R EC₅₀ = 24 nM at OX2R EC₅₀ = 24 nM) (Zhang et al., 2021). Intraperitoneal injection of RTOXA-43 increased time awake, decreased time asleep, and increased sleep/wake consolidation in 12-month old mice (Zhang et al., 2021). The effects of small molecule orexin agonists in cognitive functions have not been evaluated. In addition, further studies revealed poor metabolic stability of both YNT-185 and RTOXA-43, supporting exploration of derivatives with enhanced stability. We herein report the discovery and characterization of the dual agonist ROXA-47 with enhanced metabolic stability and its effects in improving memory, and further elucidate the roles of the two orexin receptors using selective antagonists.

2. Materials and methods

2.1. Drugs

YNT-185, ROXA-47 and derivatives were synthesized at Research Triangle Institute (RTI international), Research Triangle Park, NC, following procedures as described in the supplemental materials (purity >98%). For the in vivo experiments, 40 mg/kg of ROXA-47 and YNT-185 in saline (i.p.) were injected via a small-gauge 3/10CC insulin syringe during the light cycle (immediately after the training sessions). The agonist dose was selected based on the earlier sleep/wake sleep studies using the OX2R agonist YNT-185 and dual OXR agonist RTOXA-43 in mice (Irukayama-Tomobe et al., 2017; Zhang et al., 2021). The OX1R (SB334867) and OX2R (JNJ-10397049) antagonists were administered 15 min prior to the injection of agonists at a dose of 5 mg/kg, IP (Alijanpour et al., 2019; Bian et al., 2020).

2.2. OX1R and OX2R calcium mobilization assays

Activity of all compounds at the human OX1R and OX2R receptors was determined utilizing CHO RD-HGA16 cells (Molecular Devices) engineered to stably express either the human OX1R or the human OX2R. Cells were maintained in Ham's F12 supplemented with 10% fetal bovine serum, 100 units of penicillin and streptomycin, and 100 μg/ml normocin^™. To conduct the assay, cells were plated at 25,000 cells/well into 96-well black wall/clear bottom assay plates and incubated overnight at 37 °C, 5% CO₂. The next day prior to the assay, Calcium 5 dye (Molecular Devices) was reconstituted according to the manufacturer's instructions. The reconstituted dye was diluted 1:40 in pre-warmed (37 °C) assay buffer (1× HBSS, 20 mM HEPES, 2.5 mM probenecid, 0.25% BSA, pH 7.4 at 37 °C). The growth medium was removed and the cells were gently washed with 100 μl of pre-warmed (37 °C) assay buffer. The cells were incubated for 45 min at 37 °C, 5% CO₂ in 200 μl of the diluted Calcium 5 dye. Serial dilutions of test compounds and the agonist control orexin-A were prepared at 10× the desired final concentration in 1% DMSO/assay buffer, aliquoted into 96-well polypropylene plates, and warmed to 37 °C for 15 min. After the dye-loading incubation period, cells were pretreated with 25 μl of 9% DMSO/assay buffer and incubated for 15 min at 37 °C. After the pre-treatment incubation period, the plate was read with a FlexStation II (Molecular Devices). Calcium-mediated changes in fluorescence were monitored every 1.52 s over a 60 s time period, with the FlexStation II adding 25 μl of the test compound/control serial dilutions at the 19 s time point (excitation at 485 nm, detection at 525 nm). Peak kinetic reduction (SoftMax, Molecular Devices) relative fluorescent units (RFU) were plotted against the log of compound concentration and nonlinear regression analysis was used to generate EC₅₀ values (GraphPad Prism, GraphPad Software, Inc., San Diego, CA).

2.3. ADME studies

2.3.1. LC-MS/MS method used to quantify ROXA-47 concentrations

ROXA-47 bioanalytical samples were analyzed by LC-MS/MS using a CTC PAL autosampler, Thermo Accela UPLC and a Thermo Quantum triple quadrupole mass spectrometer. The [M + H]⁺ adduct of ROXA-47 and internal standards were monitored using positive mode electrospray ionization in multiple reaction monitoring (MRM) mode with precursor ion of 341.0 and product ion of 128.0. The lower and upper limits of quantification are 0.25 ng/ml and 1000 ng/ml respectively. The analytes were injected onto a C18 column and chromatographed using a reverse phase gradient with 0.1% formic acid in water and 0.1% formic acid in isopropanol/acetonitrile (1:4). Peak integrations were performed using Thermo Xcalibur (v2.4) software.

2.3.2. Metabolic stability

Metabolic stability assessment was performed by Paraza Pharma Inc. (Montreal, Canada). Compounds were incubated with rat liver microsomes at 37 °C for a total of 45 min. The reaction was performed at pH 7.4 in 100 mM potassium phosphate buffer containing 0.5 mg/ml of rat liver microsomal protein. Phase I metabolism was assessed by adding NADPH to a final concentration of 1 mM and collecting samples at time points 0, 5, 15, 30 and 45 min. All collected samples were quenched 1:1 with ice-cold stop solution (1 μM labetalol and 1 μM glyburide in acetonitrile) and centrifuged to remove precipitated protein. Resulting supernatants were further diluted 1:4 with acetonitrile:water (1:1). Samples were analyzed by LC/MS/MS and calculations for half-life, and in vitro clearance were accomplished using Microsoft Excel (2007). Half-life and clearance were determined from two independent experiments in duplicate. Peak areas were determined from extracted ion chromatograms. The slope value, k, was determined by linear regression of the natural logarithm of the remaining percentage of the parent drug vs. incubation time curve. The in vitro half-life (in vitro t1/2) was determined from the slope value:

$$\mathrm{in}\mathrm{vitro}{\mathrm{t}}_{3/2}=-\left(0.693/\mathrm{k}\right)$$

Conversion of the in vitro t1/2 (min) into the in vitro intrinsic clearance (in vitro CLint, in μl/min/mg protein) was done using the following equation (mean of duplicate determinations):

$$\mathrm{in}\mathrm{vitro}{\mathrm{CL}}_{\mathrm{int}}=\frac{0.693}{{\mathrm{t}}_{\frac{1}{2}}}\times \frac{\mathrm{volume}\mathrm{of}\mathrm{incubation}\left(\mu \mathrm{t}\right)}{\mathrm{amount}\mathrm{of}\mathrm{proteins}}\left(\mathrm{mg}\right)$$

2.3.3. Kinetic solubility

The kinetic solubility of the test compounds was measured in HCl at pH = 2.0. A 10 μl of test compound stock solution (20 mM DMSO) was combined with 490 μl of PBS buffer to reach a targeted concentration of 400 μM. The solution was agitated on a VX-2500 multi-tube vortexer (VWR) for 2 h at room temperature. Following agitation, the sample was filtrated on a glass-fiber filter (1 μm) and the eluate was diluted 400-fold with a mixture of acetonitrile: water (1:1). On each experimental occasion, nicardipine and imipramine were assessed as reference compounds for low and high solubility, respectively. All samples were assessed in triplicate and analyzed by LC-MS/MS using electrospray ionization against standards prepared in the same matrix.

2.4. Receptor selectivity determination

The target selectivity of ROXA-47 was assessed in radioligand binding assays against a panel of ~50 GPCRs, ion channels, and transporters at a single concentration of 10 μM by the NIMH Psychoactive Drug Screening Program (PDSP) according to their standard protocols. These targets included adrenergic (α_1A, α_1B, α_1D, α_2A, α_2B, α_2C, β1, β2, β3), dopamine (D1 - D5), GABA_A, histamine (H1–H4), muscarinic (M1–M5), opioid (μ, κ, δ), serotonin (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT6, 5-HT7A), sigma (σ1, σ2), dopamine transporter (DAT), norepinephrine transporter (NET), serotonin transporter (SERT), and BZP Rat Brain Site. The percentage of inhibition was the average of two determinations. When significant displacement of radioligand was observed (>50% inhibition at 10 μM), complete concentration-dependent displacement curves (in duplicate) were constructed to generate IC₅₀ values. IC₅₀ values were determined by a nonlinear regression analysis. The equilibrium dissociation constant (Ki) was calculated with the Cheng–Prusoff equation using the observed IC₅₀ of ROXA-47, the concentration of radioligand, and the historical values of K_d of the ligand. The full methods and references can be found at: http://pdspdb.unc.edu/pdspWeb.

2.5. Animals

All experimental procedures in this study were approved by the Minneapolis VA Health Care System Animal Care and Use Committee. Twelve-mo. old and 6-mo. old wild type mice were used for this study. The mice were obtained from the breeding colony at the Minneapolis VA Health Care System, which were bred on a wild-type C57BL/6 J background (Jackson Laboratories, Bar Harbor, ME USA). Mice were maintained in group housing (or single housing, during behavioral testing) under a 12:12-h light-dark (lights on at 0600 h) cycle in a temperature-controlled room at 21–22 °C. Teklad 8604 Lab Chow (Harlan, Indianapolis, IN USA) (3.1 kcal/g; 18.6% protein; 6.2% fat; 44% carbohydrates; Harlan Teklad 8604) and water were provided ad libitum.

2.6. Two-way active avoidance (TWAA) task

Twelve mo. old and 6 mo. old male and female mice were used for this study. Mice were transferred to the automated two-way shuttle box (size, 45.7 × 20.3 × 30.5 cm; model 75-FSFX-Fusion; AccuScan Instruments, Columbus, OH USA) and allowed 5 min of acclimation time. During acclimation and training trials, mice could move freely throughout the shuttle box. We trained mice with a 20-trial shuttle box two-way active avoidance task. The procedures for the conditioned and unconditioned stimulus are described in our earlier publications (Noble et al., 2014; Mavanji et al., 2017) and were as follows: a tone (5000 Hz; 45 dB) and a pulsatile light (2.5 Hz) were presented as a conditioned stimulus in the compartment with the mice, which was paired 5 s later with a scrambled foot shock (unconditioned stimulus, 0.3 mA) delivered through the floor grid (steel rods 0.5 cm in diameter, spaced 1.5 cm between centers). The “scrambled” nature of the shock prevents the animals from finding a “no-shock” position on the floor. To avoid receiving a foot shock the mouse had 5 s to move to the opposite compartment. If the animal did not move to the other compartment, the unconditioned stimulus was delivered for a maximum of 7 s, and the conditioned stimulus ended with the unconditioned stimulus. While receiving the unconditioned stimulus, if the animal moved to the other compartment, both conditioned and unconditioned stimuli ended immediately. The inter-trial interval was variable with a mean of 40 s. A computer using remote monitoring system software (Fusion software, AccuScan Instruments, Columbus, OH USA) was used to control experimental protocols and data collection (Noble et al., 2014; Mavanji et al., 2017). For studies involving agonist treatment, immediately following TWAA baseline training as described above, mice were injected one time with either intraperitoneal saline or OX agonists, then tested for TWAA response (20 trials) 24 h later without additional treatment. The cognitive performance tests were performed 24-h after the agonist administration with the goal of testing the effect of agonists on memory consolidation, rather than acquisition. Performance was analyzed for avoidances (crossing after conditioned stimulus, but before shock delivery), escapes (crossing to opposite side during shock delivery), total responses (avoidances + escapes), intertrial crossings (a measure of locomotor function) and escape latency (interval between conditioned stimulus and a response).

2.7. Contextual object recognition

For the contextual object recognition task (CORT), two contexts with different physical features were used. This consists of a blue square chamber (40 × 40 × 40 cm, context 1), and a grey circular arena made of metal (30 cm in height × 45 cm in diameter, context 2). For both contexts, some irregular figures made of cardboard were placed on the walls to serve as spatial landmarks. Two types of objects (A and B; for example, white glass bulbs and transparent glass jars) were used for this task. All objects were fixed to the floor at the back corners of the arena (10 cm from walls) to prevent them from being displaced by the mice. To avoid olfactory cues, objects were thoroughly cleaned with 70% ethanol prior to testing the first mouse, between mice, and after. The CORT was performed as described in our previous publication with slight modifications (Stanojlovic et al., 2019a), and we used both male and female mice for this study. The procedure lasted 4days and consisted of 3 phases: day 1 = habituation, day 2 = sample phase 1, day 3 = sample phase 2, and day 4 = test phase. On day 1, animals were habituated to both contexts (90 min apart) without any objects for 3 min, and then were handled for 1 min. On day 2, mice were placed in one of the arenas (context 1) facing the wall opposite the objects and were allowed to explore two identical objects (A1 and A2) for 10 min. On day 3, sample phase 2 was conducted (24 h after the sample phase 1), and mice were placed in context 2 together with two identical objects (which are different from the objects presented on the previous day (i.e., B1 and B2)), and were allowed to explore the objects for 10 min. Immediately after training on day 3, animals were injected one time with either intraperitoneal saline or OX agonists. The memory test was performed 24 h later, on day 4, in which mice were reintroduced to context 2 and allowed to explore freely for 3 min, with the presence of one copy of the previously presented object in context 2 (B3) together with a copy of one of the objects previously presented in context 1 but not presented in context 2 (A3). Context recognition indices (CRI) were calculated as follows: time of exploration of familiar object in novel context/(time of exploration of familiar object in familiar context + time of exploration of familiar object in novel context). Exploration was considered as pointing the nose towards an object at less than 1 cm and/or touching it with the nose. Turning around or sitting on the objects was not considered as exploratory behavior. A recognition index equal to 0.5 reflects no preference for any of the objects. An index higher than 0.5 shows a preference for familiar objects in novel contexts. The animals were able to distinguish between the familiar object in the matching context and the familiar object in the non-matching context resulting in more exploratory behavior towards the object that is placed in the non-matching environment.

2.8. Drug injections

Prior to drug injections, animals were acclimated to the restraint and injection procedure with three days of IP saline infusion. The infusions of either saline or orexin agonists (RTI International, Research Triangle Park, NC) or orexin antagonists (Tocris Bioscience, Minneapolis, MN) were made immediately after training either in the TWAA or CORT. The animals were gently scruffed to place and injectate was delivered IP. All injections occurred between 1000 and 1130 h.

2.9. Statistical analyses

All data were analyzed using either Prism 6.0 (GraphPad Software, CA) or SPSS (IBM, NY). For the experiment with both 6 mo. old and 12 mo. old animals, a two-factor (age x treatment) ANOVA, followed by the Sidak or Bonferroni post-hoc analysis for multiple comparisons. Statistical analyses of all other study data were performed using an independent-samples t-test. Significance was considered to be achieved where p < 0.05. Analyses were performed to determine the differences in retention between treatment groups. Statistics were performed using GraphPad Prism.

3. Results

3.1. Potency of ROXA-47 and derivatives

The potency (EC₅₀) of all compounds was determined in a calcium mobilization assay using CHO RD-HGA16 cells (Molecular Devices) engineered to stably express either the human OX2R or OX1R as previously described (German et al., 2013; Zhang et al., 2021). As shown in Table 1, compounds 1–10 were inactive at concentrations up to 10 μM. Compounds 11–14 showed moderate to good potency at both receptors. In particular, compound 14 (ROXA-47) had EC₅₀'s of 16 nM and 21 nM at OX1R and OX2R, respectively.

Table 1.

Potency of ROXA-47 and derivatives at OX1R and OX2R.

No.	R	X	OX1Ra		OX2Ra
			EC50 (nM)	Emax (%)	EC50 (nM)	Emax (%)
1	Me		>10,000		>10,000
2			>10,000		>10,000
3	Me		>10,000		>10,000
4			>10,000		>10,000
5	Me		>10,000		>10,000
6			>10,000		>10,000
7	Me		>10,000		>10,000
8			>10,000		1450 ±600	81 ± 8b
9	Me		>10,000		>10,000
10			>10,000		>10,000
11	Me		3080 ±190	120 ± 5	919 ± 250	107 ± 6b
12	Me		2000 ± 470	95 ± 1	385 ± 60	82 ± 1
13	Me		403 ± 80	103 ± 1	406 ± 43	78 ± 1
14 (ROXA-47)			16 ± 3	92 ± 1	21 ± 3	99 ± 2

^aEC₅₀ and E_max values (% of orexin-A control) are the means ± SEM. of at least three independent experiments conducted in duplicate. EC₅₀ values >10,000 nM were generated from two independent experiments conducted in duplicate.

^bConcentration-response curve does not have a top plateau.

3.2. ADME studies

ROXA-47, together with YNT-185 and RTOXA-43, were evaluated for their metabolic stability and kinetic solubility. In rat liver microsomes, YNT-185 and RTOXA-43 showed a half-life (t_1/2) of 0.5 min and 1.9 min, respectively. ROXA-47 showed a t_1/2 of 39.7 min, with clearance of 35.1 μl/min/mg (Table 2). All 3 compounds showed excellent solubility at pH of 2.0, the physiological pH in the stomach.

Table 2.

ADME properties of YNT-185, RTOXA-43 and ROXA-47.

	Rat liver microsome		Kinetic solubility (μM)2
	t1/2 (min)	Clearance (μl/min/mg protein)
YNT-185	0.5	2585.7	≥400.01
RTOXA-43	1.9	719.5	313.0
ROXA-47	39.7	35.1	≥400.01

¹All values were within 20% of the nominal value.

²Testing was conducted at pH = 2.0.

3.3. Receptor selectivity

ROXA-47 at 10 μM showed <50% inhibition of most of the GPCRs, enzymes or transporters tested, except 5HT₂C (87.85% inhibition) (see Supplemental Materials, Table 1S), although it had an IC₅₀ < 5 determined by binding using complete concentration-dependent displacement curves, suggesting minimal activity at this receptor. YNT-185 at 10 μM showed <50% inhibition against most of the targets, and moderate activity at peripheral benzodiazepine receptor (PBR, pKi = 6.00) and Sigma 1 receptor (pKi = 5.60).

3.4. ROXA-47 improves TWAA memory in normally aging 12 mo. old male mice

We tested performance of 12 mo. old and 6 mo. old male mice in a HC-dependent two-way active avoidance (TWAA) task. As shown in Fig. 1, the 12 mo. old male animals exhibited memory impairments as indicated by significantly [F = 98.54 (1,28), P < 0.001] higher latency (Fig. 1A) and lower [F = 41.36 (1,28), P < 0.001] total response (Fig. 1B) during the test performed 24 h after the training. Importantly, the 12 mo. old mice were moving within the same compartment as the shock, in response to the stimulus, rather than crossing to the opposite compartment, indicating absence of TWAA memory. The 12 mo. old mice treated with vehicle (saline) showed significant impairments in TWAA learning when tested 24 h after the training (higher response latency, and reduced total response (avoidance + escapes, Fig. 1)). A 40 mg/kg, i.p. dose of ROXA-47 in the12 mo. old and 6 mo. old mice produced a lower latency [F = 29.73 (1,28), P < 0.001] than the corresponding saline-treated groups (Fig. 1A). The effect was more pronounced in the 12 mo. old mice than 6 mo. old mice. Similarly, the total response (escapes + avoidances) was lower in saline-treated 12 mo. old mice compared to saline-treated 6 mo. old mice (Fig. 1B). However, ROXA-47-treated 12 mo. old mice had a significantly [F = 9.84 (1,28), P < 0.01] higher total response than that of saline-treated 12 mo. old mice, similar to the latency measurement. In 6 mo. old mice, although the total response was comparable, ROXA-47-treated mice had significantly [F = 6.10 (1,28), P < 0.05] more avoidances (Fig. 1D) and less escapes [F = 6.10 (1,28), P < 0.05] (Fig. 1C) than saline-treated mice. Finally, ROXA-47-treated 12 mo. old mice had a similar total response compared to ROXA-47-treated (and saline-treated) 6 mo. old mice. However, the ROXA-47-treated 12 mo. old mice had significantly more escapes [F = 14.19 (1,28), P < 0.05] and a non-significant (P = 0.07) increase in avoidances (Fig. 1C and D).

Fig. 1.

Improved cognition in 6 mo. old and (12 mo. old male mice as tested with TWAA), following peripheral injection of ROXA-47 (40 mg/kg, i.p.) or saline (n = 8/group *p < 0.05, **p < 0.01 ***p < 0.005).

3.5. OX1R mediates the ROXA-47 effect on TWAA memory in 12 mo. old male mice

Studies thus far suggest OX1R plays a more prominent role in HC-dependent memory and OX1R blockade would attenuate the memory enhancing effects of orexin agonists (Akbari et al., 2006; Akbari et al., 2008; Zhao et al., 2014; Gao et al., 2023). To determine the roles of OX1R and OX2R, OX1R (SB-334867) or OX2R (JNJ-10397049) antagonists (5 mg/kg, i.p.) were injected into 12-mo. old male mice 15 min prior to ROXA-47 (40 mg/kg, i.p.) injections. All injections were performed immediately after training in TWAA and animals were tested 24 h after the training. Like the results depicted in Fig. 2, ROXA-47-treated mice had a significantly [F = 11.29 (3,20), P < 0.001] reduced latency (Fig. 2A) and higher total responses [F = 7.02 (3,20), P < 0.01, Fig. 2B]. Similarly, ROXA-47 significantly enhanced the number of escapes [F = 12.03 (3,20), P < 0.001], and tended to increase the number of avoidances, which reached significance only in the group treated with both the agonist and the OX2R antagonist (JNJ-1039704, Fig. 2E (JNJ + ROXA-47)). SB-334867 blocked ROXA-47-induced enhancement of TWAA memory by increasing the latency and decreasing the total responses (which is due to a significant reduction in the number of escapes Fig. 2D). However, JNJ-10397049 did not alter the enhancement by ROXA-47, suggesting that the ROXA-47 improves cognitive function in mice primarily via OX1R activation. Interestingly, the agonist or the antagonists did not alter the number of inter-trial crossings (moving between compartments during the inter-trial interval, also a measure of locomotor activity), indicating that these compounds did not alter general locomotor activity in the tested mice (Fig. 2C).

Fig. 2.

Improved cognition in 12-mo. old male mice in TWAA observed by ROXA-47 is blocked by OX1R antagonist SB-334867, but not OX2R antagonist JNJ-10397049. A. Latency to a response, B. Total responses, C. Inter-trial crossings, D. # of escapes and E. # of avoidances. (n = 6/group, *p < 0.05, **p < 0.01 and ***p < 0.001).

3.6. Antagonist alone treatment does not affect cognition in the TWAA task

To determine if antagonist alone treatment alters TWAA performance in 12 mo. old male mice, OX1R (SB-334867) or OX2R (JNJ-10397049) antagonists (5 mg/kg, i.p.) were injected into these mice without agonist injection. All injections were performed immediately after training in TWAA and animals were tested 24 h after the training. As expected, ROXA-47-treated mice had a significantly [F = 3.5 (3,24), P < 0.05] reduced latency (Fig. 3A) and higher total responses [F = 15.80 (3,24), P < 0.001, Fig. 3B]. Similarly, ROXA-47 significantly enhanced the number of escapes [F = 7.5 (3,24), P < 0.01, Fig. 3D], and number of avoidances [F = 8.02 (3,24), P < 0.001, Fig. 3E]. Treatment with either SB-334867 or JNJ-10397049 alone did not alter TWAA performance compared to vehicle treatment, suggesting that antagonist alone treatment does not later TWAA performance in 12 mo. old mice. Similarly, the antagonists when injected alone without agonists, did not alter the number of inter-trial crossings, indicating that these compounds do not alter general locomotor activity in mice (Fig. 3C).

Fig. 3.

Antagonist alone treatment does not affect cognition in the TWAA task. A. Latency to a response, B. Total responses, C. Inter-trial crossings, D. # of escapes and E. # of avoidances. OX1R antagonist SB-334867 (SB); OX2R antagonist JNJ-10397049397049 (JNJ). (n = 6/group, *p < 0.05, **p < 0.01 and ***p < 0.001).

3.7. ROXA-47 improves TWAA memory in normally aging 12 mo. old female mice

After observing improved TWAA performance in male mice following the agonist treatment, we tested performance of 12 mo. old and 6 mo. old female mice in TWAA task. As shown in Fig. 4, a 40 mg/kg, i.p. dose of ROXA-47 in the 6 mo. old and 12 mo. old mice produced a lower latency [P < 0.01] than the corresponding saline-treated groups (Fig. 4A & F, 6 mo. & 12 mo. old respectively). The ROXA-47-treated 6 mo. and 12 mo. old mice had a significantly [P < 0.01] higher total response than that of saline-treated mice of the same age groups (Figs. 4B & G). Similarly, the ROXA-47-treated mice of both age groups had significantly [P < 0.05] more escapes (Fig. 4D & I) than saline-treated mice. However, the number of inter-trial crossings and avoidances are identicel between ROXA-47 and saline treated mice of both age groups.

Fig. 4.

Improved cognition in 6 mo. old and 12 mo. old female mice as tested with TWAA, following peripheral injection of ROXA-47 (40 mg/kg, i.p.) or saline (n = 6/group *p < 0.05, **p < 0.01).

3.8. YNT-185 improves TWAA memory in mice

We tested performance of 12 mo. old male and 6 mo. old female mice in TWAA task following the administration of OX2R agonist YNT-185. As shown in Fig. 5, a 40 mg/kg, i.p. dose of YNT-185 in the 6 mo. old female and 12 mo. old male mice produced a lower latency [P < 0.01] than the corresponding saline-treated groups (Fig. 5A & F, 6 mo. females & 12 mo. old rmales espectively). The YNT-185 treated mice had a significantly [P < 0.05] higher total response than that of saline-treated mice of the same age group and sex (Figs. 5B & G). Similarly, the YNT-185 treated mice of both sex at respective age groups had significantly [P < 0.05] more escapes (Fig. 5D & I) than saline-treated mice. However, the number of inter-trial crossings and avoidances are identicel between YNT-185 and saline treated mice of same sex at the respective age.

Fig. 5.

Improved cognition in 6-mo. old female and 12-mo. old male mice as tested with TWAA, following peripheral injection of YNT-185 (40 mg/kg, i.p.) or saline (n = 6/group *p < 0.05, **p < 0.01).

3.9. ROXA-47 and YNT-185 improves CORT memory in normally aging 12 mo. old female mice

ROXA-47 was then evaluated in another memory model, CORT, together with the reported OX2R agonist YNT-185. Intraperitoneal administration of the OX2R agonist YNT-185 in 12 mo. old female mice significantly (P < 0.01) improved memory as indicated by enhancement of CRI following the injection (Fig. 6A). Like the YNT-185 administration, ROXA-47-treatment significantly (P < 0.01) enhanced memory in these mice immediately after training by elevating their CRI compared to saline injection (Fig. 6B). Similarly, ROXA-47-treatment significantly (P < 0.01) enhanced CRI in 6 mo. old female mice compared to saline injection (Fig. 6B). Interestingly, the CRI index was significantly (P < 0.05) lower in 12 old vs. 6 mo. old female mice (0.24 vs. 0.45), indicating an aging induced cognitive decline. These results show that i.p. administered YNT-185 or ROXA-47 improves HC-dependent cognitive function in mice, similar to that of centrally administered orexin-A (Mavanji et al., 2017; Stanojlovic et al., 2019a).

Fig. 6.

Improved context recognition index in 12-mo. old female mice following peripheral injection of YNT-185 (40 mg/kg, i.p. A), or ROXA-47 in 12-mo. old (40 mg/kg, i.p. B) and 6 mo.-old female mice (40 mg/kg, i.p. C) compared to saline treatment. n = 6 & 8 for 6-mo. and 12-mo. groups respectively *p < 0.05 and **p < 0.0.01.

3.10. ROXA-47 improves CORT memory in normally aging 12 mo. old male mice

We then evaluated the effect of ROXA-47 on CORT performance in male mice. Intraperitoneal administration of ROXA-47 in 6 mo. old (Fig. 7A), and 12 mo. old male mice (Fig. 7B) significantly (P < 0.01) improved memory as indicated by enhancement of CRI following the injection compared to saline injection. Interestingly, the CRI index was significantly (P < 0.05) lower in 12 old vs. 6 mo. old male mice (0.13 vs. 0.37), indicating an aging induced cognitive decline. These results show that i.p. administered ROXA-47 improves HC-dependent cognitive function in male mice.

Fig. 7.

Improved context recognition index in 6 mo. old and 12-mo. old male mice following peripheral injection ROXA-47 compared to saline treatment. A. CI after saline or ROXA-47 treatment in 6 mo. old male mice. B. CI after saline or ROXA-47 treatment in 12 mo. old male mice (40 mg/kg, i.p.). n = 6 for both 6-mo. and 12-mo. groups *p < 0.05.

3.11. OX1R mediates the ROXA-47 effect on CORT memory in mice

To determine the roles of OX1R and OX2R, OX1R (SB-334867) or OX2R (JNJ-10397049) antagonists (5 mg/kg, i.p.) were injected into 6-mo. old female mice and 12 mo. old male mice 15 min prior to ROXA-47 (40 mg/kg, i.p.) injections. All injections were performed immediately after training in CORT and animals were tested 24 h after the training. ROXA-47-treated 6 mo. old female mice had a significantly [F = 3.006 (5,30), P < 0.05] higher CRI (Fig. 8A). Similarly, ROXA-47 significantly enhanced the CRI in 12 mo. old male mice [F = 2.65 (5,27), P < 0.05, Fig. 8B]. SB-334867 blocked ROXA-47-induced enhancement of CORT memory by decreasing the CRI in both groups. However, JNJ-10397049 did not alter the enhancement by ROXA-47, suggesting that the ROXA-47 improves cognitive function in mice primarily via OX1R activation. Interestingly, the antagonists alone did not alter the CORT performance in both groups, indicating that these compounds did not alter CORT memory when given in the absence of agonists.

Fig. 8.

Improved context recognition index observed after ROXA-47 is blocked by OX1R antagonist SB-334867, but not OX2R antagonist JNJ-10397049 in 6-mo. old (A) female, and 12-mo. old male mice (B). (n = 6/group, *p < 0.05, **p < 0.01).

4. Discussion

Orexin-A is a neuromodulator that integrates numerous physiological processes, including promotion of wakefulness, energy expenditure, learning/memory, and stabilization of sleep/wake states. The HC is a brain region involved in cognition and memory, and the neuropeptide orexin has been shown to enhance HC-dependent learning and memory (Yang et al., 2013; Mavanji et al., 2017; Stanojlovic et al., 2019a). However, the effect of small molecule orexin agonists on learning and memory have not yet been characterized. In the present study, we synthesized a more metabolically stable small molecule OX agonist, ROXA-47, and tested its effects, together with YNT-185, on hippocampal-dependent learning and memory by evaluating TWAA and CORT performance in 12-mo. old mice. Our results indicate that the dual orexin agonist ROXA-47 has a positive influence on TWAA and CORT memory in 12-mo. old mice. In addition, we showed that ROXA-47-induced TWAA and CORT memory enhancement is attenuated by pre-treatment with the OX1R antagonist SB334867, but not with pre-treatment of the OX2R antagonist JNJ-10397049, indicating that ROXA-47 enhances memory via activation of OX1R. Finally, we found that the OX2R agonist YNT-185 improves CORT performance in 12 mo. old mice. These data confirm that ROXA-47 and YNT-185 improve cognitive function in orexin-deficient 12-mo. old mice.

In the in vitro studies, both YNT-185 and the dual agonist RTOXA-43 previously reported by us showed poor metabolic stability when tested against rat liver microsomes (RLM). Our analysis suggests that the electron rich central phenyl ring likely contributes to the instability, which may be readily oxidized in physiological environments (Smith, 2010; Silverman, 2012). Therefore, derivatives with the phenyl group replaced with other groups were explored. Unfortunately, all of the non-aromatic groups examined resulted in a total loss of activity, suggesting requirement of an aromatic moiety at this site. Therefore, pyridinyl groups, which are aromatic but of lower electron density, were then explored. Excitingly, these pyridinyl derivatives showed good potency, particularly ROXA-47, at both OX1R and OX2R (Table 1). In these derivatives, the substitution on the amide group (R as shown in Table 1) was either a methyl (as in YNT) or a pyridinylmethyl (as in ROXA-47). Importantly, compared to RTOXA-43 and YNT-185, both of which showed short half-lives and high clearance, the metabolic stability of ROXA-47 was considerably enhanced, with a t_1/2 of 39.7 min and clearance of 35.1 μl/min/mg protein in RLM. These results suggest that the replacement of the central phenyl group with a less electron rich pyridinyl group successfully enhanced the metabolic stability.

The current in vivo findings with ROXA-47 are in agreement with earlier gain/loss of function studies, which showed improved cognitive function following orexin-A administration in animals (Deadwyler et al., 2007; Yang et al., 2013; Aitta-Aho et al., 2016; Chen et al., 2016). Orexin neurons project to several brain structures critical for cognitive function, including the HC and basal forebrain area, and orexin release in these areas is involved in modulating memory (Akbari et al., 2007; Yang et al., 2013; Feng et al., 2025). In orexin knockout mice, spatial working memory was impaired as measured with the delayed non-matching-to-place T-maze task and object exploration tasks, highlighting the role of orexin-A in HC-dependent memory (Dang et al., 2018). Intraventricular administration of orexin-A reversed the spatial memory impairments observed in mice subjected to chronic intermittent hypoxia (Zhu et al., 2021) and in a rat model of post-traumatic stress disorder (Han et al., 2020). In orexin/ataxin (O/A3) mice, orexin-A injection into the CA1 and dentate gyrus regions of the HC improves long-term potentiation (a cellular mechanism underlying learning and memory) (Yang et al., 2013; Zhao et al., 2014). Earlier studies from our group have demonstrated that orexin modulates HC-dependent TWAA, along with spatial and CORT memories (Mavanji et al., 2017; Stanojlovic et al., 2019a). Specifically, we demonstrated that central orexin administration rescues HC-dependent cognitive deficits in the orexin-deficient O/A3 transgenic mice (Mavanji et al., 2017). In addition, in a transgenic mouse model of Parkinson's disease (A53T mice), we demonstrated that intra-hippocampal injections of orexin or chemogenetic activation of orexin neurons ameliorated HC-dependent Barnes maze and CORT memory impairments (Stanojlovic et al., 2019a).

A previous study from our laboratory demonstrated that orexin is neuroprotective (Butterick et al., 2012), and others have shown orexin neuron loss during progression of several neurodegenerative diseases including narcolepsy, Parkinson's, Alzheimer's, and Huntington's disease (Petersen et al., 2005; Fronczek et al., 2007; Thannickal et al., 2007; Fronczek et al., 2012). We showed that mice with progressive orexin neuron loss are more prone to develop cognitive deficits than wild type animals (Mavanji et al., 2017). Interestingly, while orexin/ataxin (O/A3) and normally aging mice exhibit decreased orexin neuron density and orexin expression (Stanojlovic et al., 2019b), the expression of orexin receptors in the HC is unchanged (Mavanji et al., 2017). In addition, activation of orexin neurons restored physical activity in middle-aged mice, indicating that the orexin receptors remain intact during age progression, and these animals are responsive to exogenous orexin stimulation (Mavanji et al., 2017; Stanojlovic et al., 2019b). In the current study, we for the first time demonstrate that small molecule orexin agonists improve HC-dependent cognitive function in 12-mo. old mice, with a behavioral profile like that observed after central orexin-A injection or chemogenetic activation of orexin neurons (Mavanji et al., 2017; Stanojlovic et al., 2019a). Together, these data suggest that peripheral small molecule orexin agonists have the potential to modulate cognitive function.

Our finding that ROXA-47-induced TWAA and CORT memory enhancement is attenuated by pre-treatment with the OX1R antagonist SB334867 aligns with the prominent role of OX1R in memory. Most studies thus far suggest that OX1R plays a more prominent role in both HC- and PFC-dependent memory processing. For example, high levels of OX1R expression are found in areas critical for cognition, such as the septohippocampal nucleus, paraventricular thalamic nucleus, CA1, CA2 regions of the HC, amygdalohippocampal area and PFC (Trivedi et al., 1998; Karimi et al., 2022). This is consistent with a study suggesting orexin-A produces excitatory effects on HC neurons via OX1R (Chen et al., 2017). Our team also showed that injection of orexin-A into the CA1 region of the mouse HC improves HC-dependent memory (Mavanji et al., 2017; Stanojlovic et al., 2019a). On the other hand, microinjection of OX1R antagonist SB-334867 in hippocampal CA1 region impairs passive avoidance and spatial memory processing in rats (Akbari et al., 2007; Akbari et al., 2008). Similarly, the OX1R antagonist SB-334867 worsened the impairments in short-term working memory, long-term spatial memory, and synaptic plasticity in mouse models of Alzheimer's disease (3xTg-AD mice) (Elahdadi Salmani et al., 2022; Gao et al., 2023). Similarly, AAV-mediated knockdown of OX1R in the HC impaired spatial memory in mice (Soler et al., 2021). Moreover, the memory enhancing effect of intra-cranial self-stimulation is mediated by OX1R, as an OX1R antagonist attenuated intra-cranial self-stimulation-induced memory enhancement (García-Brito et al., 2020). Additionally, a study showed that micropressure administration of orexin-A significantly increases the spontaneous firing rate of the CA1 neurons in rats, whereas this effect is blocked by the OX1R antagonist SB-334867, indicating that orexin modulates the firing activity of CA1 neurons via OX1R (Chen et al., 2017). Furthermore, blockade of OX1Rs in the PFC impairs executive functions such as reward-based decision making (Karimi et al., 2019; Karimi et al., 2022). The SB-334867 orexin antagonist also disrupts attentional performance in rats (Boschen et al., 2009), and significantly exaggerated learning and memory loss in capsaicin-treated rats (Kooshki et al., 2017).

Interestingly, the OX2R agonist YNT-185 also improved performance in CORT and TWAA, similarly to that after ROXA-47, suggesting a possible role of OX2R in memory. The OX2R is found in the CA3 regions and dentate gyrus of the HC (Trivedi et al., 1998), regions important for learning and memory. Orexin-A at sub-nanomolar concentrations prevented the induction of depotentiation induced by low frequency stimulation and restored long-term potentiation (Selbach et al., 2010), which was blocked by OX1R, OX2R or dual orexin receptor antagonists (Lu et al., 2016). The OX2R antagonist TCSOX229 shortened the freezing time during a contextual fear test, a proven model of extinction of aversive memories (Flores et al., 2014). While it was reported as an OX2R selective agonist, studies from us and others have found that YNT-185 has considerable OX1R activity. Our calcium mobilization assays showed that YNT-185 had EC50 = 824 ± 56 nM at OX1R and EC50 = 165 ± 43 nM at OX2R. In these assays, orexin-A had EC₅₀'s = 0.6 nM and 0.3 nM at OX2R and OX1R, respectively, consistent with previous reports (Nagahara et al., 2015). Similarly, another group found that Nag 26, a close derivative of YNT-185, only had 17.6 ± 5.4-fold (n = 7) higher potency for OX2R than for OX1R in stimulating Ca²⁺ response (Rinne et al., 2018). Therefore, the presence of OX1R activity may have contributed, at least in part, to the observed memory enhancement by YNT-185. In addition, the effect of YNT-185 on memory might be indirect by the promotion of wake, which showed enhancement of wake following IP administration (Irukayama-Tomobe et al., 2017).

One possible mechanism by which agonists exert cognition promotion would be activation of the Gs pathway and downstream enhancement of cyclic-AMP response element binding protein (CREB) phosphorylation, as phosphorylated CREB (pCREB) is important for synaptic plasticity and memory consolidation (Asok et al., 2019). Earlier studies have demonstrated orexin activation of cAMP/protein kinase A (PKA)/CREB signaling pathway, formation of pCREB, long-term potentiation and promotion of long-term memory formation in rodents (Yang et al., 2013; Hou et al., 2022). Orexin deficiency is associated with lower levels of pCREB, and long-term potentiation in mice (Yang et al., 2013). In addition, activation of the orexin system also leads to stimulation of protein kinase A, C, phospholipase C (PLC, which increases intracellular calcium) and calcium calmodulin pathways which are also suggested to be involved in neuroplasticity (Chen et al., 2017; Dustrude et al., 2018; Zhu et al., 2021; Hou et al., 2022). It is also possible that agonists might improve memory through induction of neurogenesis in the dentate gyrus, as earlier studies have demonstrated induction of HC neurogenesis in rodents by orexin (Ito et al., 2008; Zhao et al., 2014; Chieffi et al., 2017). Another mechanism would be orexin-induced prevention of oxidative stress and apoptotic cell death in the HC (Zhu et al., 2021). It was also suggested that activation of the PLC pathway and increased intracellular calcium following OXR activation is involved in excitation of HC-CA1 neurons, ultimately resulting in enhanced cognitive function.

Twelve-mo. old mice exhibit decreased orexin neuron density and orexin expression (Stanojlovic et al., 2019b), yet activation of orexin neurons restored physical activity in these mice, indicating that the orexin receptors remain intact during age progression, and these receptors respond to exogenous orexin stimulation (Stanojlovic et al., 2019b). This study, together with the current results indicate that exogenous orexin agonists can rescue dormant receptor-mediated circuits, when endogenous orexin tone is low.

It should be noted there are several limitations in the current study. The current study used male mice for the TWAA studies involving orexin-antagonists, removing the ability to test for potential sex differences in antagonist action related to TWAA. Future studies will assess the effect of sex in the antagonist action on TWAA. We used a single dose of the agonist in two cognitive tests (TWAA and CORT), mainly because of the limited amount available from a multiple-step synthesis, and the dose of agonist was based on earlier published studies (Irukayama-Tomobe et al., 2017; Zhang et al., 2021). We plan to assess the dose range of orexin-agonist action and in additional cognitive tests, when ROXA-47 is available in larger quantities. Since small molecule OX1R agonists are not yet available, we were limited to testing the dual orexin-receptor agonist ROXA-47 and the OX2R agonist YNT-18 in the cognitive tests, although the experiment using dual agonist (ROXA-47) with OX2R selective antagonist (JNJ-10397049) provided early indications on the potential actions of OX1R agonists. During the CORT, the control 12 mo. old (saline-treated) groups showed a trend towards a preference for the familiar object/familiar context relative to the familiar object/novel context. Ideally, the animals would show identical preferences to both objects, but the age of these mice might have resulted in them showing a preference for the familiar object/familiar context, indicating a cognitive/behavioral impairment. While the use of a smaller sample size (n = 6–8) for the behavioral tests could impact generalizability, the robustness and significance of the behavioral effects of the agonist suggests the sample size is sufficient.

In conclusion, the present study demonstrates that increasing orexin receptor signaling by stimulation with a small molecule orexin agonist enhances memory in 12-mo. old mice. Twelve-mo. old animals were impaired in HC-dependent memory processing, and a single injection of the agonist immediately after training rescued this memory deficit. Our study also supports that orexin agonists improve memory by acting via OX1Rs, although additional experiments may be needed to rule out OX2R contributions. Together these data indicate that small molecule OX1R agonist treatment could be a potential therapeutic avenue for reversal of cognitive deficits in conditions associated with orexin loss such as aging and neurodegenerative diseases.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.pbb.2026.174246.

Data availability

The authors declare that all the data supporting the findings of this study are available within the paper and its Supplemental Data and available upon request.