TAK-994, a Novel Orally Available Brain-Penetrant Orexin 2 Receptor-Selective Agonist, Suppresses Fragmentation of Wakefulness and Cataplexy-Like Episodes in Mouse Models of Narcolepsy [Drug Discovery and Translational Medicine]

Abstract

Loss of orexin neurons is associated with narcolepsy type 1 (NT1), which is characterized by multiple symptoms including excessive daytime sleepiness and cataplexy. Orexin 2 receptor (OX2R) knockout (KO) mice, but not orexin 1 receptor (OX1R) KO mice, show narcolepsy-like phenotypes, thus OX2R agonists are potentially promising for treating NT1. In fact, in early proof-of-concept studies, intravenous infusion of danavorexton, an OX2R-selective agonist, significantly increased wakefulness in individuals with NT1. However, danavorexton has limited oral availability. Here, we report pharmacological characteristics of a novel OX2R agonist, TAK-994 [N-methanesulfonamide sesquihydrate]. TAK-994 activated recombinant human OX2R (EC50 value of 19 nM) with > 700-fold selectivity against OX1R and activated OX2R-downstream signaling similar to those by orexin peptides in vitro. Oral administration of TAK-994 promoted wakefulness in normal mice but not in OX2R KO mice. TAK-994 also ameliorated narcolepsy-like symptoms in two mouse models of narcolepsy: orexin/ataxin-3 mice and orexin-tTA;TetO diphtheria toxin A mice. The wake-promoting effects of TAK-994 in orexin/ataxin-3 mice were maintained after chronic dosing for 14 days. These data suggest that overall in vitro and in vivo properties, except oral availability, are very similar between TAK-994 and danavorexton. Preclinical characteristics of TAK-994 shown here, together with upcoming clinical study results, can improve our understanding for orally available OX2R agonists as new therapeutic drugs for NT1 and other hypersomnia disorders.

SIGNIFICANCE STATEMENT Narcolepsy type 1 (NT1) is caused by a loss of orexin neurons, and thus an orexin 2 receptor (OX2R) agonist is considered to address the underlying pathophysiology of NT1. Oral administration of TAK-994, a novel OX2R agonist, promoted wakefulness in normal mice, but not in OX2R knockout mice, and ameliorated fragmentation of wakefulness and cataplexy-like episodes in mouse models of narcolepsy. These findings indicate that TAK-994 is an orally available brain-penetrant OX2R-selective agonist with potential to improve narcolepsy-like symptoms.

Introduction

Orexin, also called hypocretin, is a neuropeptide that regulates sleep/wakefulness (de Lecea et al., 1998; Sakurai et al., 1998; Tsujino and Sakurai, 2009). Orexin also has biologic functions in energy homeostasis, stress control, and reward system (Tsujino and Sakurai, 2009). There are two types of orexins, orexin-A (OX-A) and orexin-B (OX-B); OX-A and OX-B originate from the proteolytic cleavage of a common precursor molecule, prepro-orexin, in orexin neurons of the lateral hypothalamus (de Lecea et al., 1998; Sakurai et al., 1998). Loss of orexin neurons is strongly linked to narcolepsy type 1 (NT1). The number of orexin neurons in individuals with NT1 was reduced by between 85% and 95% compared with normal subjects (Thannickal et al., 2000). Moreover, very low or undetectable levels of OX-A in the cerebrospinal fluid are observed in individuals with NT1 (Ripley et al., 2001).

Individuals with NT1 suffer from severe neurologic conditions characterized by excessive daytime sleepiness (EDS), cataplexy, hypnagogic/hypnopompic hallucinations, sleep paralysis, and disrupted nocturnal sleep (Scammell, 2015; Abad and Guilleminault, 2017). However, no drugs that address the underlying disease pathophysiology of NT1 are available (Abad and Guilleminault, 2017; Thorpy, 2020). Psychostimulants, such as modafinil and methylphenidate, are used for the treatment of EDS. Antidepressants, including venlafaxine and clomipramine, are used off label for treatment of cataplexy. Sodium oxybate and pitolisant have been approved in some countries for treatment of both EDS and cataplexy. However, despite these medications, individuals with NT1 report persistent daytime sleepiness and/or fatigue (Maski et al., 2017). Hence, a novel therapeutic approach could be useful for individuals with NT1.

Two postsynaptic G protein-coupled receptors (GPCRs) are specific for orexins, orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) (Sakurai et al., 1998). OX1R has one order of magnitude higher binding affinity for OX-A over OX-B, whereas OX2R has a similar affinity for both OX-A and OX-B (Sakurai et al., 1998). OX2R knockout (KO) mice show narcolepsy-like symptoms including fragmentation of sleep/wakefulness and cataplexy-like episodes, while OX1R KO mice do not show apparent behavioral abnormalities (Willie et al., 2001; Sakurai, 2007). Moreover, canines with null mutations in the OX2R gene (Hcrtr2) show marked narcolepsy-like symptoms (Lin et al., 1999). Thus, OX2R activation is anticipated to be a promising therapeutic strategy for NT1 (Irukayama-Tomobe et al., 2017). However, orexins cannot cross the blood–brain barrier (Fujiki et al., 2003). As such, nonpeptide and brain-penetrant OX2R agonists are of interest as potential treatments for NT1.

We have previously discovered danavorexton, a brain-penetrant OX2R-selective agonist (Yukitake et al., 2019). Danavorexton showed potent agonistic activity for recombinant human OX2R (EC50 value of 5.5 nM) with > 5,000-fold selectivity against OX1R in calcium mobilization assays. The OX2R-downstream signals activated by danavorexton were similar to those activated by orexin peptides in vitro. In an electrophysiological study, danavorexton activated endogenous OX2R on histaminergic neurons in the mouse tuberomammillary nucleus (TMN). Subcutaneous administration of danavorexton induced wake-promoting effects in C57BL/6J mice but not in OX2R KO mice, demonstrating its OX2R selectivity in vivo. Moreover, danavorexton (subcutaneous administration) improved narcolepsy-like symptoms, including fragmentation of wakefulness and cataplexy-like episodes, in orexin/ataxin-3 mice, an orexin neuron-ablated mouse model of NT1 (Evans et al., 2022; Ishikawa et al., 2022). Preclinical evaluation of danavorexton supported progression to clinical studies with an intravenous formulation. In a phase I study, danavorexton (intravenous infusion) significantly increased wakefulness in individuals with NT1 (ClinicalTrials.gov Identifier: NCT03332784 and NCT03748979) (Evans et al., 2022). However, danavorexton has limited oral availability (Yukitake et al., 2019; Evans et al., 2022; Ishikawa et al., 2022).

In this study, we characterized a novel OX2R agonist, TAK-994 [N-methanesulfonamide sesquihydrate]. Our data demonstrate that TAK-994 is an orally available and brain-penetrant OX2R-selective agonist with potential to improve narcolepsy-like symptoms in mouse models of narcolepsy.

Materials and MethodsEthics Statement

The care and use of the animals and the experimental protocols in this study were approved by the Institutional Animal Care and Use Committee of Takeda Pharmaceutical Company Limited.

Animals

In our previous study, the wake-promoting effects of an OX2R agonist danavorexton assessed by using male mice could predict the clinical efficacy in individuals with NT1 (Evans et al., 2022). Thus, male mice were used in this study. Male C57BL/6J mice obtained from CLEA Japan Inc. (Tokyo, Japan) were used for slice electrophysiology study (3- to 4-week-old), electroencephalogram/electromyogram (EEG/EMG) study (13- to 14-week-old), and pharmacokinetic study (9- to 10-week-old). Orexin/ataxin-3 mice with a C57BL/6J genetic background were obtained from University of Tsukuba (Hara et al., 2001). Male orexin/ataxin-3 mice and their wild-type (WT) littermate mice were bred in our laboratory and were used for EEG/EMG study and pharmacokinetic study at 25 to 31 weeks old. Male OX2R KO mice with a C57BL/6J genetic background were generated in our laboratory (Yukitake et al., 2019) and were used for EEG/EMG study at 14 weeks old. Orexin-tTA mice with a C57BL/6J genetic background were obtained from Nagoya University (Tabuchi et al., 2014). Orexin-tTA mice were crossed with TetO diphtheria toxin A (DTA) mice (B6.Cg-Tg(tetO-DTA)1Gfi/J, The Jackson Laboratory, Maine, USA) to generate orexin-tTA;TetO DTA mice (Tabuchi et al., 2014). In orexin-tTA;TetO DTA mice, rapid degeneration of orexin neurons by DTA expression occurs in the absence of doxycycline (DOX). Orexin-tTA;TetO DTA mice were fed DOX-containing chow (5TP7, Japan SLC, Inc., Shizuoka, Japan) until 6 weeks of age, and then DOX was removed from the chow (CE-2, CLEA Japan Inc.). Male orexin-tTA;TetO DTA mice at 32 to 34 weeks old were used for EEG/EMG study and pharmacokinetic study. All mice were housed under laboratory conditions (12-hour light/dark cycles) with food and water available ad libitum. After the experiments, mice were euthanized with carbon dioxide gas according to Institutional Animal Care and Use Committee policy.

Chemicals and Radiolabeled Ligands

TAK-994 and danavorexton (TAK-925) (Fujimoto et al., 2022) were synthesized by Takeda Pharmaceutical Company Limited. [3H]TAK-994 was synthesized by BioBridge K.K. (Tokyo, Japan). Modafinil was synthesized by LKT Laboratories, Inc. (Minnesota, USA). OX-A and OX-B were purchased from Peptide Institute Inc. (Osaka, Japan). [Ala11, D-Leu15]-OX-B was purchased from Tocris Bioscience (Bristol, UK). For in vitro studies, all compounds were dissolved in DMSO (Fujifilm Wako Pure Chemical Co., Osaka, Japan) and then were diluted in each experimental solution. For in vivo studies, TAK-994, danavorexton, and modafinil were suspended in 0.5% (w/v) methylcellulose (MC) in distilled water (Fujifilm Wako Pure Chemical Co.) and were administered orally to mice in a volume of 10 mL/kg of body weight.

Cell Lines and Culture

CHO-K1 cells (CCL-61, ATCC, Virginia, USA) stably expressing human OX1R or OX2R (hOX1R/CHO-K1 cells or hOX2R/CHO-K1 cells) were established as described previously (Yukitake et al., 2019). hourOX1R/CHO-K1 cells and hOX2R/CHO-K1 cells were used for a calcium mobilization assay. CHO cells were stably transfected with ProLink-tagged hOX2R and β-arrestin2, coupled to an inactive N-terminal β-galactosidase deletion mutant, to establish a receptor cell line (hOX2R/CHO-EA) as described previously (Yukitake et al., 2019). The inositol monophosphate (IP1) assay, β-arrestin recruitment assay, and evaluations of phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) were conducted in hOX2R/CHO-EA cells.

Radioligand Binding Assay

The saturation binding and association/dissociation kinetics analysis of [3H]TAK-994 to hOX2R expressing Expi293F cell membranes were conducted as described previously (Ishikawa et al., 2022). Data analysis was carried out using GraphPad Prism 6 (GraphPad software, Inc., Califonia, USA). To determine the receptor number (Bmax) and the equilibrium dissociation constant (pKD) in saturation binding studies, data were globally fitted with a nonlinear regression of one site‐total and nonspecific binding. To determine the association rate constant (kon), the dissociation rate constant (koff), and pKD values, data were globally fitted to single-phase exponential association and decay equations. Dissociation t1/2 was calculated as ln(2)/koff.

Calcium Mobilization Assay

Calcium mobilization in hOX1R/CHO-K1 cells or hOX2R/CHO-K1 cells was measured using an FDSS/μCELL system (Hamamatsu Photonics K.K., Hamamatsu, Japan) as described previously (Yukitake et al., 2019). The responses to 0.11% DMSO (hOX2R/CHO-K1 cells) or 0.5% DMSO (hOX1R/CHO-K1 cells) in the absence and presence of 100 nM OX-A were used as 0% and 100% responses, respectively.

To evaluate positive allosteric modulator activity of TAK-994, OX-A-induced calcium responses were measured in the presence of various concentrations of TAK-994 in hOX2R/CHO-K1 cells. The responses to 0.2% DMSO in the absence and presence of 100 nM OX-A were used as 0% and 100% responses, respectively.

IP1 Assay

Intracellular accumulation of IP1 in hOX2R/CHO-EA cells was measured using an IP-One Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer (HTRF) assay kit (PerkinElmer Inc., Massachusetts, USA) as described previously (Yukitake et al., 2019). The responses to 0.3% DMSO in the absence and presence of 1 μM OX-A were used as 0% and 100% responses, respectively.

β-Arrestin Recruitment Assay

The binding of β-arrestin to hOX2R in hOX2R/CHO-EA cells was assessed using a PathHunter assay kit (DiscoveRx, California, USA) as described previously (Yukitake et al., 2019). The PathHunter signals were measured using an EnVision plate reader (PerkinElmer Inc.). The responses to 0.3% DMSO in the absence and presence of 1 μM OX-A were used as 0% and 100% responses, respectively.

Evaluations of Phosphorylation of ERK1/2 and CREB

Intracellular phosphorylation of ERK1/2 (Thr202/Tyr204) and CREB (Ser133) in hOX2R/CHO-EA cells was measured using an HTRF phospho-ERK assay kit (PerkinElmer Inc.) and HTRF phospho-CREB assay kit (PerkinElmer Inc.), respectively, as described previously (Yukitake et al., 2019). HTRF signals were detected using an EnVision plate reader. The responses to 0.3% DMSO in the absence and presence of 3 μM OX-A were used as 0% and 100% responses, respectively.

In Vitro Off-Target Profiling of TAK-994

Activity of TAK-994 (10 μM) on various receptors, ion channels, and enzymes (106 targets in total) was evaluated at Eurofins Panlabs Discovery Services Taiwan, Ltd. (Taipei, Taiwan).

Slice Electrophysiology Study

Experiments were performed using slices of the posterior hypothalamus containing TMN, prepared from C57BL/6J mice as described previously (Yukitake et al., 2019). Histaminergic neurons in the TMN were identified by the presence of the transient outward current and the inwardly rectifying current activated by hyperpolarization (Kamondi and Reiner, 1991). Pharmacological effects of test compounds on the membrane potential were assessed in the presence of 1 μM tetrodotoxin to block neuron firing. Membrane potentials during the last 30 seconds of each treatment condition were averaged, and the delta values between the membrane potential and the baseline were analyzed. Recordings with large access resistance (> 30 MΩ) were abandoned. Signals were recorded using an Axopatch 700B amplifier and a Digidata 1322A digitizer board, bandpass filtered at 2 kHz, sampled at 10 kHz and analyzed with pClamp10.3 software (Molecular Devices, California, USA).

Surgery, Data Acquisition, and Vigilance State Determination in Mice

Implantation of EEG/EMG electrodes and EEG/EMG recordings were carried out as described previously (Yukitake et al., 2019). EEG/EMG signals were amplified, filtered (EEG, 0.5–250 Hz; EMG, 16–250 Hz), digitized at a sampling rate of 200 Hz, and recorded using VitalRecorder (Kissei Comtec Co., Ltd., Nagano, Japan). Locomotor activity was measured by an infrared activity sensor (Biotex, Kyoto, Japan). Wakefulness, non-rapid eye movement (NREM) sleep, or rapid eye movement (REM) sleep were automatically classified in 4-second epochs using SleepSign software (Kissei Comtec Co., Ltd.), based on EEG/EMG/locomotion data (Yukitake et al., 2019). Each stage was characterized as follows: (1) wakefulness, low-amplitude EEG with high-voltage EMG activities or locomotion score; (2) NREM sleep, high-amplitude slow-wave EEG with low-voltage EMG activities; and (3) REM sleep, theta-dominated EEG with EMG atonia. Time spent in wakefulness, NREM sleep, and REM sleep and the number and duration of wakefulness episodes were also calculated using SleepSign software.

Evaluation of Sleep/Wakefulness States in Mice

A cross-over study design with at least 2 days washout between doses was used to evaluate drug efficacy on sleep/wakefulness states in mice. For the sleep phase experiments, vehicle (i.e., 0.5% MC in distilled water) or TAK-994 was administered to C57BL/6J mice (10 or 30 mg/kg, by mouth), OX2R KO mice (30 mg/kg, p.o.), WT mice (3 or 10 mg/kg, by mouth), or orexin/ataxin-3 mice (1 or 3 mg/kg, by mouth) at zeitgeber time (ZT) 5 when mice are mostly asleep (Gotter et al., 2013), and then EEG/EMG were recorded. Vehicle or danavorexton (25 or 50 mg/kg, by mouth) was administered to C57BL/6J mice at ZT 5, and then EEG/EMG were recorded. Vehicle or modafinil (3, 10, or 30 mg/kg, by mouth) was administered to WT mice or orexin/ataxin-3 mice at ZT 5, and then EEG/EMG were recorded. For the active phase experiments, vehicle or TAK-994 was administered to orexin/ataxin-3 mice (3 or 10 mg/kg, by mouth) or orexin-tTA;TetO DTA mice (1 or 3 mg/kg, by mouth) at ZT12, and then EEG/EMG were recorded.

Evaluation of Cataplexy-Like Episodes in Mice

Murine cataplexy-like episodes were scored according to the following four criteria: (1) abrupt episode of EMG atonia lasting ≥ 20 seconds, (2) behavioral immobility during the episode (assessed by locomotion data), (3) predominance of theta activity during the episode, and (4) ≥ 40 seconds of wakefulness preceding the episode (Scammell et al., 2009). Palatable food, such as chocolate, can increase the number of cataplexy-like episodes during the active phase in orexin KO mice (Clark et al., 2009; Oishi et al., 2013). Thus, chocolate was used as a trigger of positive emotions to increase the number of cataplexy-like episodes in orexin/ataxin-3 mice. Evaluation of drug efficacy on cataplexy-like episodes was conducted with a cross-over design (at least 2 days washout between doses). Vehicle or TAK-994 (3 mg/kg, by mouth) was administered to orexin/ataxin-3 mice at ZT12, then milk chocolate (Hershey’s Kisses milk chocolate, Hershey, Pennsylvania, USA) was placed in the cage followed by EEG, EMG, and locomotor activity recordings.

Orexin-tTA;TetO DTA mice are known to have more severe cataplexy-like episodes ataxin-3/ataxin-3 mice even in the absence of chocolate (Tabuchi et al., 2014). Therefore, the effect of TAK-994 on spontaneous cataplexy-like episodes in orexin-tTA;TetO DTA mice was studied in the absence of chocolate. Vehicle or TAK-994 (1 or 3 mg/kg, by mouth) was administered to orexin-tTA;TetO DTA mice at ZT12, and then EEG, EMG, and locomotor activity were recorded.

EEG Power Spectral Analysis in Mice

EEG power spectral analysis was carried out using fast Fourier transformation of EEG data during wakefulness using SleepSign software as described previously (Ishikawa et al., 2022). EEG power was divided into five frequency bands: (1) delta (0.75–4 Hz), (2) theta (6–10 Hz), (3) alpha (10–13 Hz), (4) beta (13–30 Hz), and (5) gamma (30–80 Hz). EEG power density was expressed as the percentage of each frequency band within the accumulated power between 0.75 and 80 Hz.

Effects of Repeated Administration of TAK-994 on Sleep/Wakefulness States in Mice

Orexin/ataxin-3 mice were grouped into two cohorts: a control group and a 14-day treatment group. In the control group, the vehicle (i.e., 0.5% MC in distilled water) was administered orally to mice three times a day (ZT12, ZT15, and ZT18) for 14 days. In the 14-day treatment group, TAK-994 (30 mg/kg, by mouth) was administered to mice three times a day (ZT12, ZT15, and ZT18) for 14 days. EEG/EMG were recorded on day 1 and day 14 (active phase: ZT12–ZT21, subsequent sleep phase: ZT0–ZT10).

Pharmacokinetic Study in Mice

In C57BL/6J mice, blood samples were collected at various time points after administration of TAK-994 [10 and 30 mg/kg, by mouth (0.5, 1, 2, 4, and 8 hour)] or danavorexton [25 mg/kg, by mouth (0.25, 0.5, 1, 1.5, and 2 hour)]. Blood samples were collected from WT mice and orexin/ataxin-3 mice at 0.5, 1, and 2 hours after administration of TAK-994 (3 and 10 mg/kg, by mouth). In the repeated administration study, TAK-994 (30 mg/kg, by mouth) was administered to orexin/ataxin-3 mice in both the control group and the 14-day treatment group at ZT12, ZT15, and ZT18 on day 15, and blood samples were collected at various time points (0, 0.5, 3, 3.5, 6, 6.5, 9, and 24 hours) after the first administration of TAK-994. In orexin-tTA;TetO DTA mice, blood samples were collected at various time points (0.25, 0.5, 1, 1.5, 2, 3, 4, and 8 hours) after administration of TAK-994 (1 and 3 mg/kg, by mouth). Plasma was separated from the blood samples by centrifugation. The plasma concentrations of compounds were quantified with high-performance liquid chromatography-tandem mass spectrometry. The lower limit of quantitation was 3 ng/mL for plasma. The maximum concentration (Cmax), the time taken to reach Cmax (Tmax), area under the concentration-time curve, and mean residence time (MRT) were calculated.

Statistics

All data were presented as the mean ± S.E.M. The EC50 values were calculated by XLfit (IDBS, Massachusetts, USA) or GraphPad Prism 6 from the data expressed as percentage of control. For in vivo studies, statistical analysis was performed using EXSUS (CAC EXICARE Corporation, Tokyo, Japan). Pairwise differences between groups were evaluated using a two-tailed paired t test. In experiments with multiple doses of test compounds, the statistical differences were analyzed using a two-tailed Williams test (for parametric data) or a two-tailed Shirley–Williams test (for nonparametric data). Comparisons for time-elapsed data were analyzed using repeated measures ANOVA followed by a post hoc two-tailed Student's t test with Bonferroni correction. For all analyses, a P value of ≤ 0.05 was considered significant.

ResultsTAK-994 Selectively Binds and Activates Recombinant and Endogenous OX2R

Chemical structure of TAK-994 is shown in Fig. 1A. To characterize the binding kinetics of TAK-994 to OX2R, we performed receptor binding assays using [3H]TAK-994. [3H]TAK-994 bound to hOX2R in a monophasic manner, with pKD of 7.07 and Bmax of 4.03 pmol/mg protein (Fig. 1B). Kinetic binding studies showed that [3H]TAK-994 associated rapidly to hOX2R and dissociated rapidly from hOX2R within 5 minutes (Fig. 1C). The kon, koff, and dissociation t1/2 were 4.20 × 107 M−1 min−1, 1.28 minute−1, and 0.89 minute, respectively (Supplemental Table 1). The dissociation constant (koff/kon) calculated from the kinetics analysis (pKD = 7.47) was comparable with the value determined from the saturation binding assay (pKD = 7.07), suggesting good accuracy of these parameters.

Fig. 1.Fig. 1.Fig. 1.

TAK-994 selectively binds and activates recombinant and endogenous OX2R. (A) Chemical structure of TAK-994. (B) Saturation binding curve and Scatchard plot (inset, representative data) of [3H]TAK-994 binding to hOX2R expressing Expi293F cell membranes. Specific binding was obtained by calculating the difference between total binding and nonspecific binding measured in the presence of 100 µM unlabeled TAK-994. Mean ± S.E.M. of a single experiment performed in triplicate. (C) Kinetic binding profile of [3H]TAK-994 to hOX2R expressing cell membranes. Dissociation was initiated by adding 100 µM unlabeled TAK-994 at time 30 minutes. Mean ± S.E.M. of four independent experiments, each performed in triplicate. (D) Effect of TAK-994 on calcium mobilization in hOX2R/CHO-K1 cells and hOX1R/CHO-K1 cells. The responses to 100 nM OX-A represented the 100% response. Mean ± S.E.M., n = 4. (E) Evaluation for positive allosteric modulation activity of TAK-994 in hOX2R/CHO-K1 cells. OX-A-induced calcium responses were measured in the presence of various concentrations of TAK-994. The responses to 100 nM OX-A represented the 100% response. Mean ± S.E.M., n = 4. (F) Effects of TAK-994, OX-A, and OX-B on intracellular accumulation of IP1 in hOX2R/CHO-EA cells. The responses to 1 μM OX-A represented the 100% response. Mean ± S.E.M., n = 4. (G) Effects of TAK-994, OX-A, and OX-B on β-arrestin recruitment in hOX2R/CHO-EA cells. The responses to 1 μM OX-A represented the 100% response. Mean ± S.E.M., n = 4. (H) Effects of TAK-994, OX-A, and OX-B on intracellular phosphorylation of ERK1/2 at Thr202/Tyr204. The responses to 3 μM OX-A represented the 100% response. Mean ± S.E.M., n = 4. (I) Effects of TAK-994, OX-A, and OX-B on intracellular phosphorylation of CREB at Ser133. The responses to 3 μM OX-A represented the 100% response. Mean ± S.E.M., n = 4. (J) Representative examples of membrane potential change of histaminergic neurons in mouse TMN by [Ala11, D-Leu15]-OX-B and TAK-994. (K) Membrane potential change by [Ala11, D-Leu15]-OX-B or TAK-994. Each data point represents the average of 5 to 8 recordings using brain slices obtained from 7 to 10 mice. [Ala11, D-Leu15]-OX-B (10, 1000 nM): n = 5, [Ala11, D-Leu15]-OX-B (30, 100, 300 nM): n = 7. TAK-994 (3 nM): n = 7, TAK-994 (10, 30, 100, 300, 1000 nM): n = 8. Mean ± S.E.M.

Next, we characterized OX2R-selective agonistic activity of TAK-994 using hOX2R/CHO-K1 cells and hOX1R/CHO-K1 cells. TAK-994 increased calcium mobilization in hOX2R/CHO-K1 cells in a dose-dependent manner with an EC50 value of 19 nM (Fig. 1D). On the contrary, EC50 value of TAK-994 in calcium mobilization using hOX1R/CHO-K1 cells was 14,000 nM (Fig. 1D). Therefore, TAK-994 had > 700-fold OX2R selectivity over OX1R. TAK-994 did not affect the EC50 values of OX-A in calcium mobilization assay using hOX2R/CHO-K1 cells (Fig. 1E and Supplemental Table 2), which indicated that TAK-994 is an orthosteric, full agonist for OX2R with no positive allosteric modulation activity. The inhibitory or stimulatory activities of TAK-994 against various enzymes, receptors, and ion channels (106 targets in total) were assessed. TAK-994 at 10 μM did not induce more than 50% inhibition or stimulation of any enzymes, receptors, and ion channels, except progesterone receptor B (55% inhibition), indicating high selectivity of TAK-994 for OX2R with minimal off-target activity in vitro (Supplemental Tables 3 and 4).

OX2R signaling is mediated by several G-proteins (Gq, Gs, and Gi proteins) as well as other proteins such as β-arrestin (Sakurai et al., 1998; Zhu et al., 2003; Dalrymple et al., 2011). Activation of the Gq protein-coupled receptor leads to generation of inositol 1,4,5-trisphosphate, which is quickly degraded to IP1. Thus, we examined the effect of TAK-994 on IP1 production in hOX2R/CHO-EA cells using orexin peptides as controls. In hOX2R/CHO-EA cells, OX-A, OX-B, and TAK-994 dose-dependently increased IP1 contents with EC50 values of 16, 36, and 37 nM, respectively (Fig. 1F). We next evaluated β-arrestin recruitment in hOX2R/CHO-EA cells (Dalrymple et al., 2011). OX-A, OX-B, and TAK-994 increased β-arrestin recruitment with EC50 values of 4.5, 6.6, and 100 nM, respectively (Fig. 1G). OX2R activation by orexin peptides has been reported to induce ERK1/2 and CREB phosphorylation via protein kinase C signaling in OX2R-expressed CHO cells (Guo and Feng, 2012). OX-A, OX-B, and TAK-994 induced phosphorylation of ERK1/2 with EC50 values of 19, 32, and 170 nM, respectively (Fig. 1H) and phosphorylation of CREB with EC50 values of 2.9, 5.0, and 39 nM, respectively (Fig. 1I), in hOX2R/CHO-EA cells. These results suggest that TAK-994 activated OX2R downstream signals similar to those activated by orexin peptides.

Next, we assessed the effect of TAK-994 on endogenous OX2R expressed on histaminergic neurons in the mouse TMN using whole-cell patch-clamp method (Yukitake et al., 2019). Histaminergic neurons in the TMN receive projections from orexin neurons in the hypothalamus (Marcus et al., 2001). OX2R, but not OX1R, is highly expressed in histaminergic neurons (Marcus et al., 2001; Mieda et al., 2011); therefore, activation of OX2R can be assessed by measuring the membrane potential of these cells. [Ala11, D-Leu15]-OX-B, an OX-B peptide analog with 400-fold selectivity for OX2R over OX1R, was used as a control (Asahi et al., 2003). The whole-cell patch-clamp recording indicated that both [Ala11, D-Leu15]-OX-B and TAK-994 induced dose-dependent depolarization of membrane potential in histaminergic neurons in the mouse TMN with EC50 values of 102.6 and 19.7 nM, respectively (Fig. 1, J and K). These results suggest that TAK-994 activates endogenous OX2R in a similar manner with orexin peptides.

Oral Administration of TAK-994 Produces Wake-Promoting Effects via OX2R Activation at Threefold Lower Dosage in NT1 Model Mice Compared with WT Mice during the Sleep Phase

We previously reported that subcutaneous administration of danavorexton at 1 and 3 mg/kg significantly promoted wakefulness in C57BL/6J mice during the sleep phase (Yukitake et al., 2019). To determine if oral administration is also available as a route of administration of danavorexton, effect of danavorexton (by mouth) on wakefulness was evaluated in C57BL/6J mice during the sleep phase (Supplemental Fig. 1A). Danavorexton even at a high dose of 25 mg/kg, by mouth showed limited plasma exposure for less than 1 hour after administration in C57BL/6J mice (Tmax: 0.5 hour; MRT: approximately 0.5 hour) (Supplemental Fig. 1B and Supplemental Table 5). As a result, danavorexton (by mouth) did not produce significant arousal effects up to 50 mg/kg in C57BL/6J mice (Supplemental Fig. 1, C–E). These results suggest that danavorexton has low oral bioavailability in mice.

To characterize the in vivo profile of TAK-994 after oral administration, we assessed the effect of TAK-994 on sleep/wakefulness states in C57BL/6J mice and OX2R KO mice during the sleep phase. TAK-994 was administered orally to C57BL/6J mice and OX2R KO mice at ZT5, and then sleep/wakefulness states were monitored by EEG/EMG recordings (Fig. 2A). By considering the pharmacokinetic profile of TAK-994 in C57BL/6J mice (Tmax: < 1 hour; MRT: approximately 1 hour; Supplemental Fig. 2 and Supplemental Table 6), efficacy of TAK-994 was evaluated during the first hour after administration. In C57BL/6J mice, TAK-994 (30 mg/kg, by mouth) significantly increased total wakefulness time (Fig. 2B), accompanied by a significant decrease in total NREM sleep time and no change in total REM sleep time (Supplemental Fig. 3, A and B). On the contrary, TAK-994 (30 mg/kg, by mouth) did not affect total wakefulness time in OX2R KO mice (Fig. 2C). Thus, TAK-994 has good oral bioavailability and brain penetration and exerts arousal effects through activation of OX2R in mice.

Fig. 2.Fig. 2.Fig. 2.

Oral administration of TAK-994 produces wake-promoting effects via OX2R activation at threefold lower dosage in NT1 model mice compared with WT mice during the sleep phase. (A) Time schedule of drug administration in mice during the sleep phase. TAK-994 was administered orally to mice at ZT5 and then EEG/EMG were recorded. Analysis was performed with data collected during the first hour after drug administration. (B) Effect of TAK-994 (10 and 30 mg/kg, by mouth) on wakefulness time in C57BL/6J mice (n = 8). (C) Effect of TAK-994 (30 mg/kg, by mouth) on wakefulness time in OX2R KO mice (n = 6). (D) Effect of TAK-994 (3 and 10 mg/kg, by mouth) on wakefulness time in WT mice (n = 6). (E) Effect of TAK-994 (1 and 3 mg/kg, by mouth) on wakefulness time in orexin/ataxin-3 mice (n = 6). Mean ± S.E.M. *P ≤ 0.05, ***P ≤ 0.001, compared with the vehicle-treated mice (two-tailed Williams test). n.s., not significant by two-tailed paired t test.

After a long period of orexin loss, NT1 model mice may cause changes in sensitivity to TAK-994 as was seen in danavorexton (Evans et al., 2022). To explore this possibility, we evaluated the wake-promoting effects of TAK-994 in orexin/ataxin-3 mice and their littermate WT mice. TAK-994 was administered orally to WT mice and orexin/ataxin-3 mice at ZT5, and then EEG/EMG were recorded (Fig. 2A). Pharmacokinetic profiles of TAK-994 in the plasma were comparable in WT mice and orexin/ataxin-3 mice (Supplemental Fig. 4 and Supplemental Table 7). In WT mice, TAK-994 (10 mg/kg, by mouth) significantly increased total wakefulness time (Fig. 2D), accompanied by decreases in total NREM sleep time and REM sleep time (Supplemental Fig. 5, A and B). By considering possible sensitivity difference, 3 mg/kg was used as a higher dose of TAK-994 in orexin/ataxin-3 mice. In orexin/ataxin-3 mice, TAK-994 (3 mg/kg, by mouth) significantly increased total wakefulness time (Fig. 2E), decreased total NREM sleep time, and did not affect total REM sleep time (Supplemental Fig. 5, C and D). These results indicate that orexin/ataxin-3 mice have threefold higher sensitivity to TAK-994 compared with WT mice in wake-promoting effects.

Modafinil is the first-line treatment of EDS in NT1 (Golicki et al., 2010; Barateau et al., 2016). Although its precise mechanism of action is unknown, dopaminergic and noradrenergic systems are prime targets for the action of modafinil (Gerrard and Malcolm, 2007). We compared the sensitivity of WT mice and orexin/ataxin-3 mice to wake-promoting effects of modafinil. Modafinil was administered to mice at ZT5, and then EEG/EMG were recorded (Supplemental Fig. 6A). In WT mice, modafinil (30 mg/kg, by mouth) significantly increased total wakefulness time during the first hour after administration, accompanied by a significant decrease in total NREM sleep time and a tendency to decrease in total REM sleep time (Supplemental Fig. 6B). In orexin/ataxin-3 mice, modafinil (30 mg/kg, by mouth) significantly increased total wakefulness time, decreased total NREM sleep time, and did not affect total REM sleep time (Supplemental Fig. 6C). These results indicate that modafinil increased wakefulness in both WT mice and orexin/ataxin-3 mice at the same minimum effective dose (i.e., 30 mg/kg). Taken together, higher sensitivity in orexin/ataxin-3 mice compared with WT mice may be specific to OX2R agonists. OX2R system may be sensitized to OX2R ligand after the long-term loss of orexin in orexin/ataxin-3 mice.

TAK-994 Ameliorates Narcolepsy-Like Symptoms in Orexin/Ataxin-3 Mice during the Active Phase

Orexin/ataxin-3 mice show narcolepsy-like symptoms such as fragmentation of sleep/wakefulness and cataplexy-like episodes (Hara et al., 2001). We examined the effects of TAK-994 on these symptoms in orexin/ataxin-3 mice after administration at the onset of the active period (ZT12) (Fig. 3A). Figure 3B shows representative hypnograms after administration of TAK-994 (3 mg/kg, by mouth). During the first hour after administration, TAK-994 (3 and 10 mg/kg, by mouth) significantly increased total wakefulness time (Fig. 3C) and decreased total NREM sleep time and REM sleep time (Supplemental Fig. 7, A and B). The wake-promoting effects of TAK-994 (10 mg/kg, by mouth) were maintained for about 2 hours after administration (Supplemental Fig. 7C). TAK-994 also significantly ameliorated fragmentation of wakefulness with fewer number of wakefulness episodes (Fig. 3D) and longer mean duration of wakefulness episodes (Fig. 3E). EEG power spectral analysis is widely used to investigate the drug properties associated with functional modulation of brain in clinical and preclinical studies (Lin et al., 2008; Conrado et al., 2013). Evaluation of the effect of TAK-994 on EEG power density during wakefulness in orexin/ataxin-3 mice revealed that TAK-994 (10 mg/kg, by mouth) significantly increased gamma power during wakefulness (Fig. 3F).

Fig. 3.Fig. 3.Fig. 3.

TAK-994 ameliorates narcolepsy-like symptoms in orexin/ataxin-3 mice during the active phase. (A) Time schedule of drug administration in orexin/ataxin-3 mice during the active phase (evaluation for sleep/wakefulness states). TAK-994 was administered orally to mice at ZT12, and then EEG/EMG were recorded. Analysis was performed with data collected during the first hour after drug administration. (B) Representative hypnogram for 2 hours after TAK-994 (3 mg/kg, by mouth) administration in orexin/ataxin-3 mice. Effect of TAK-994 (3 and 10 mg/kg, by mouth) on wakefulness time (C), number of wakefulness episodes (D), and duration of wakefulness episode (E) in orexin/ataxin-3 mice during the active phase. (F) Effect of TAK-994 on EEG power density during wakefulness in orexin/ataxin-3 mice. (G) Time schedule of drug administration in orexin/ataxin-3 mice during the active phase (evaluation for cataplexy-like episodes). TAK-994 was administered orally to mice at ZT12, and then chocolate was placed in the cage. The number of cataplexy-like episodes was determined during 3 hours after drug administration. (H) Effect of TAK-994 (3 mg/kg, by mouth) on cataplexy-like episodes in orexin/ataxin-3 mice during the active phase. Mean ± S.E.M., n = 7. **P ≤ 0.01, ***P ≤ 0.001, compared with the vehicle-treated mice (two-tailed Williams test). #P ≤ 0.05, ##P ≤ 0.01, compared with the vehicle-treated mice (two-tailed Shirley–Williams test). $P ≤ 0.05, compared with the vehicle-treated mice (two-tailed paired t test).

Next, we examined the effect of TAK-994 on cataplexy-like episodes in orexin/ataxin-3 mice during the active phase. We performed this study in the presence of chocolate as a rewarding stimulus to increase the frequency of cataplexy (Fig. 3G) (Clark et al., 2009; Oishi et al., 2013). In the presence of chocolate, cataplexy-like episodes were approximately 1 episode per hour; therefore, we decided to evaluate the number of cataplexy-like episodes during 3 hours after TAK-994 administration (at ZT12). TAK-994 (3 mg/kg, by mouth) significantly suppressed cataplexy-like episodes in orexin/ataxin-3 mice (Fig. 3H). These results suggest that TAK-994 ameliorates multiple narcolepsy-like symptoms such as fragmentation of wakefulness and cataplexy-like episodes in orexin/ataxin-3 mice during the active phase.

TAK-994 Ameliorates Narcolepsy-Like Symptoms in Orexin-tTA;TetO DTA Mice during the Active Phase

Characterization of efficacy of TAK-994 in multiple animal models of narcolepsy can enhance the confidence for the translatability of our preclinical findings. Thus, we used another narcolepsy mouse model, orexin-tTA;TetO DTA mice, to further evaluate the effect of TAK-994 on narcolepsy-like symptoms. TAK-994 was administered orally to orexin-tTA;TetO DTA mice at ZT12, and then EEG/EMG were recorded to assess sleep/wakefulness states (Fig. 4A). Pharmacokinetic data of TAK-994 (1 and 3 mg/kg, by mouth) in orexin-tTA;TetO DTA mice are shown in Supplemental Fig. 8 and Supplemental Table 8. Pharmacokinetic profiles after administration of TAK-994 (3 mg/kg, by mouth) in orexin/ataxin-3 mice and orexin-tTA;TetO DTA mice were comparable (Cmax in the plasma, 805.1 and 805.4 ng/mL; and Tmax of the plasma, 0.5 and 0.42 hours, in orexin/ataxin-3 mice and orexin-tTA;TetO DTA mice, respectively) (Supplemental Table 7 and 8). As shown in the representative hypnograms, TAK-994 (3 mg/kg, by mouth) appeared to improve sleep/wakefulness fragmentation in orexin-tTA;TetO DTA mice (Fig. 4B). Detailed analysis of the EEG/EMG data revealed that TAK-994 (3 mg/kg, by mouth) significantly increased total wakefulness time during the first hour after administration (Fig. 4C), accompanied by decreases in total NREM sleep time and REM sleep time (

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