TAK-861, a potent, orally available orexin receptor 2-selective agonist, produces wakefulness in monkeys and improves narcolepsy-like phenotypes in mouse models

Abstract

Narcolepsy type 1 (NT1) is associated with severe loss of orexin neurons and characterized by symptoms including excessive daytime sleepiness and cataplexy. Current medications indicated for NT1 often show limited efficacy, not addressing the full spectrum of symptoms, demonstrating a need for novel drugs. We discovered a parenteral orexin receptor 2 (OX2R) agonist, danavorexton, and an orally available OX2R agonist, TAK-994; both improving NT1 phenotypes in mouse models and individuals with NT1. However, danavorexton has limited oral availability and TAK-994 has a risk of off-target liver toxicity. To avoid off-target-based adverse events, a highly potent molecule with low effective dose is preferred. Here, we show that a novel OX2R-selective agonist, TAK-861 [N-{(2S,3R)-4,4-Difluoro-1-(2-hydroxy-2-methylpropanoyl)-2-[(2,3',5'-trifluoro[1,1'-biphenyl]-3-yl)methyl]pyrrolidin-3-yl}ethanesulfonamide], activates OX2R with a half-maximal effective concentration of 2.5 nM and promotes wakefulness at 1 mg/kg in mice and monkeys, suggesting ~ tenfold higher potency and lower effective dosage than TAK-994. Similar to TAK-994, TAK-861 substantially ameliorates wakefulness fragmentation and cataplexy-like episodes in orexin/ataxin-3 and orexin-tTA;TetO DTA mice (NT1 mouse models). Compared with modafinil, TAK-861 induces highly correlated brain-wide neuronal activation in orexin-tTA;TetO DTA mice, suggesting efficient wake-promoting effects. Thus, TAK-861 has potential as an effective treatment for individuals with hypersomnia disorders including narcolepsy, potentially with a favorable safety profile.

Keywords: Danavorexton; Narcolepsy type 1; Orexin; Orexin receptor 2 agonist; TAK-861; TAK-994.

Fig. 1

Activation of recombinant and physiological OX2R by TAK-861. (a) Chemical structure of TAK-861. (b) Effect of TAK-861 on calcium mobilization in hOX2R/CHO-K1 cells (left) and hOX1R/CHO-K1 cells (right). The responses to 100 nM OX-A represented the 100% response. Mean ± SD; n = 4. (c) Representative examples of membrane potential changes of histaminergic neurons in mouse tuberomammillary nucleus by TAK-861. (d) Dose–response changes in membrane potential by TAK-861. Each data point represents the average of six to eight recordings using brain slices obtained from seven mice. Mean ± standard error of the mean. (e) Effect of TAK-861 on intracellular accumulation of IP1 in hOX2R/CHO-EA cells. OX-A and OX-B were used as controls. The responses to 1 μM OX-A represented the 100% response. Mean ± SD; n = 4. (f) Effect of TAK-861 on β-arrestin recruitment in hOX2R/CHO-EA cells. OX-A and OX-B were used as controls. The responses to 1 μM OX-A represented the 100% response. Mean ± SD; n = 4. (g) Effect of TAK-861 on intracellular phosphorylation of ERK1/2 at Thr202/Tyr204. OX-A and OX-B were used as controls. The responses to 1 μM OX-A represented the 100% response. Mean ± SD; n = 4. (h) Effect of TAK-861 on intracellular phosphorylation of CREB at Ser133. OX-A and OX-B were used as controls. The responses to 1 μM OX-A represented the 100% response. Mean ± SD; n = 4. CHO-K1 Chinese hamster ovary cells, CREB cAMP response element-binding protein, ERK1/2 extracellular signal-regulated kinase 1/2, hOX1R human OX1R, hOX2R human OX2R, IP1 inositol monophosphate, OX-A orexin-A, OX-B orexin-B, OX1R orexin receptor 1, OX2R orexin receptor 2, SD standard deviation.

Fig. 2

Wake-promoting effects of TAK-861 in WT mice, cynomolgus monkeys, and NT1 model mice during the sleep phase. (a) Time schedule of drug administration in mice during the sleep phase. TAK-861 or vehicle was administered orally to mice at ZT5, and then EEG/EMG and locomotor activity were recorded. (b) Effect of TAK-861 at 0.3, 1, and 3 mg/kg on wakefulness time in 10-min bins for 3 h (left) and total wakefulness time for 1 h (right) after administration in WT mice. Mean ± SEM; n = 8. **p < 0.01, ***p < 0.001, compared with the vehicle-treated mice, as determined by two-tailed Shirley-Williams test. (c) Effect of TAK-861 at 10 mg/kg on wakefulness time in WT mice (n = 7) and OX2R KO mice (n = 8). ***p < 0.001, compared with vehicle-treated mice, as determined by two-tailed paired t-test. (d) Effect of TAK-861 at 0.03, 0.1, and 0.3 mg/kg on wakefulness time in orexin/ataxin-3 mice. Mean ± SEM; n = 8. ***p < 0.001, compared with vehicle-treated mice, as determined by two-tailed Williams test. (e) Quantification of [³H]-EMPA binding in brain regions (arrows) in the autoradiograms of orexin/ataxin-3 mice and WT littermates (N = 6–8). The statistical analysis was performed between orexin/ataxin-3 mice and WT littermates using an analysis of variance (ANOVA) for repeated measures followed by post hoc two-tailed Student’s t-test with Bonferroni correction. ***p < 0.001. (f) Time schedule of drug administration in cynomolgus monkeys during the sleep phase. TAK-861 or vehicle was administered orally to mice at ZT12, and then EEG/EMG and locomotor activity were recorded. (g) Effects of TAK-861 on wakefulness time at 0.3 (left) and 1 mg/kg (middle) in 1-h bins for 8 h and total wakefulness time for 8 h (right) after administration in cynomolgus monkeys. Mean ± SEM; n = 8. Left and middle: *p < 0.05, **p < 0.01, ***p < 0.001, compared with the vehicle-treated mice, as determined by repeated measures analysis of variance followed by a post hoc two-tailed paired t-test, with multiplicity adjusted using the Holm method. Right: ***p < 0.001, compared with vehicle-treated cynomolgus monkeys in each group, as determined by paired t-test. AHA anterior hypothalamic, AHip amygdalohippocampal area, Ctx-Int internal layer of cortex, EEG electroencephalogram, EMG electromyogram, Hip-CA Cornu Ammonis (CA) of the hippocampus, Hip-DG dentate gyrus of the hippocampus, KO knockout, MHA medial hypothalamic area, NAc nucleus accumbens, NT1 narcolepsy type 1, Pont-nu pontine nuclei, SEM standard error of the mean, Sup-coll superior colliculus, WT wild type, ZT zeitgeber time.

Fig. 3

Effects of TAK-861 on 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 for evaluation for sleep/wakefulness states. TAK-861 or vehicle was administered orally to mice at ZT12, and then EEG/EMG and locomotor activity were recorded. (b) Representative hypnogram for 3 h after TAK-861 (1 mg/kg) or vehicle administration in orexin/ataxin-3 mice. (c) Effect of TAK-861 (0.1, 0.3, and 1 mg/kg) on wakefulness time in 10-min bins for 3 h (left) and total wakefulness time for 1 h (right) after administration in orexin/ataxin-3 mice. Effect of TAK-861 (0.1, 0.3, and 1 mg/kg) on (d) number of wakefulness episode and (e) mean duration of wakefulness episode for 1 h after administration in orexin/ataxin-3 mice. Mean ± SEM; n = 8. **p < 0.01, ***p < 0.001, compared with vehicle-treated mice, as determined by two-tailed Shirley-Williams test. (f) Time schedule of drug administration in orexin/ataxin-3 mice during the active phase for evaluation of cataplexy-like episodes. After drug administration, chocolate was placed in the cage. The number of cataplexy-like episodes was determined for 3 h after administration. (g) Effect of TAK-861 (0.1, 0.3, and 1 mg/kg) on cataplexy-like episodes in orexin/ataxin-3 mice. Mean ± SEM; n = 8. **p < 0.01, compared with vehicle-treated mice, as determined by two-tailed Shirley-Williams test. (h) Time schedule of repeated drug administration in mice during the active phase. In the control or sub-chronic treatment group, vehicle or TAK-861 was orally administered to mice at ZT12 for 14 days, respectively. In the acute treatment group, vehicle was orally administered to mice at ZT12 for 13 days then TAK-861 was administrated on day 14. (i) Effects of acute and sub-chronic treatment of TAK-861 (1 mg/kg) on wakefulness time in 1-h bins for 12 h (left) and total wakefulness time for 1 h (right) after administration in orexin/ataxin-3 mice. Mean ± SEM; n = 8. ***p < 0.001, compared with vehicle-treated mice, as determined by Tukey’s multiple comparison test. n.s. not significant, EEG electroencephalogram, EMG electromyogram, SEM standard error of the mean, ZT zeitgeber time.

Fig. 4

Effects of TAK-861, modafinil, and clomipramine on narcolepsy-like symptoms in orexin-tTA;TetO DTA mice during the active phase. (a) Time schedule of drug administration in orexin-tTA;TetO DTA mice during the active phase for evaluation for sleep/wakefulness states and cataplexy-like episodes. TAK-861, modafinil, clomipramine, or vehicle was administered orally to mice at ZT14, and then EEG/EMG and locomotor activity were recorded. Effects on wakefulness time of (b) TAK-861 (0.3 and 1 mg/kg), (c) modafinil (30 and 100 mg/kg), and (d) clomipramine (15 and 50 mg/kg) for 3 h after administration in orexin-tTA;TetO DTA mice. Blue arrows indicate the sampling time for the c-Fos mapping study in Fig. 5. Effects on cataplexy-like episodes of (e) TAK-861 (0.3 and 1 mg/kg), (f) modafinil (30 and 100 mg/kg), and (g) clomipramine (15 and 50 mg/kg) for 3 h after administration in orexin-tTA;TetO DTA mice. Mean ± standard error of the mean; n = 7–8. *p < 0.05, **p < 0.01, ***p < 0.001, compared with vehicle-treated mice, as determined by two-tailed Williams/Shirley-Williams test. DTA diphtheria toxin A, EEG electroencephalogram, EMG electromyogram.

Fig. 5

Brain-wide neuronal activity induced by TAK-861, modafinil, and clomipramine in orexin-tTA;TetO DTA mice during the active phase. (a) The heatmap shows significant changes (q < 0.05) of the c-Fos expression with the direction of z-score comparing with vehicle in the 747 regions of interest. The color bar shows the 33 brain categories and corresponding colors. The four pairwise correlation heatmaps were generated using Pearson’s correlation coefficients (− 1 to 1) from inter-regional c-Fos expression data across subjects for each group; (b) vehicle, (c) TAK-861, (d) modafinil, and (e) clomipramine. ACA anterior cingulate area, AI agranular insular area, AUD auditory areas, CB cerebellum, CTXsp cortical subplate, DTA diphtheria toxin A, ECT ectorhinal area, ENT entorhinal area, FRP frontal pole cerebral cortex, GU gustatory areas, HIP hippocampal region, HY hypothalamus, ILA infralimbic area, MB midbrain, MO somatomotor areas, MY medulla, OLF olfactory areas, ORB orbital area, P pons, PAL pallidum, PAR para-subiculum, PERI perirhinal area, PL prelimbic area, POST post-subiculum, PRE pre-subiculum, PTLp posterior parietal association areas, RSP retrosplenial area, SS somatosensory areas, STR striatum, SUB subiculum, TEa temporal association areas, TH thalamus, VISC visceral area, VIS visual areas.