OX₁ ( IC50 = 1789 nM ); OX₂ ( IC50 = 18 nM ); OX₁ ( Ki = 1584 nM ); OX₂ ( Ki = 0.5 nM )

MK-1064 (30 mg/kg, oral administration) electively induces sleep via OX2R in wild-type mice in rodents[2]. MK-1064 (30 mg/kg, oral administration, 5 days) reverses the struggle behavior that rats pretreated with CNO had developed[3]. MK-1064 (1-5 mg/kg, intravenous injection/oral administration) exhibits moderate oral bioavailability and clearance in rat, dog, and rhesus monkey[1].

MK-1064, a selective OX2R antagonist, (Roecker et al., 2014) was dissolved overnight in 20% Vitamin E d-a-tocopherol polyethylene glycol 1000 succinate (Vit E-TPGS). Rats were orally administered either 20% Vit E-TPGS as vehicle or 30 mg/kg MK-1064, both at 1 mL/kg for consistent volume. All animals were given a vehicle dose 1 day prior to the start of the restraint paradigm to habituate to the oral gavage procedure. Animals received injections of vehicle (20% Vit E TPGS, p.o.) or MK-1064 and vehicle (saline and 8% DMSO, i.p.) or CNO 90 min prior to the start of the 30-min restraint. This timing was chosen based on the fact that both MK-1064 and CNO have been shown to promote behavioral effects in the rat within 30 min of administration and effects last up to 4 h after administration (Alexander et al., 2009; Farrell and Roth, 2013; Hasegawa et al., 2014; Roecker et al., 2014). Additionally, CNO was given at the time of MK-1064 injection to minimize the effects of repeated handling prior to restraint.

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Assessing HPA reactivity to acute or repeated restraint with orexin manipulations[3]
A naïve cohort of rats injected with DREADD-containing virus was exposed to either 1 or 5 consecutive days of restraint with injections of vehicle or CNO IP and either vehicle or MK-1064 administered PO 90 min prior to each restraint. See Fig. 3A for depiction of experimental paradigm. Animals were weighed on Day 1 and Day 5 of restraint. Video cameras were set up above the restrainers in order to record struggle behavior on day 2 of restraint. While rats are in the restrainer for 30 min, most of the struggle behavior occurs within the first 10 min. Therefore, we analyzed struggle behavior during this time period. A trained investigator blind to experimental groups hand scored struggle behavior – defined as attempts to escape, or intense movement of the animal while in the restrainer. Blood was collected on Day 1 and Day 5 of restraint to assess the HPA response to acute and repeated restraint, respectively. Briefly, on day 1, tail blood was taken at 0 min (prior to being placed in restraint), again at 15 min and 30 min (during restraint), and at 60 min (recovery time point in the home cage). Plasma corticosterone and Adrenocorticotropic Hormone (ACTH) were assayed with a Radioimmunoassay kit from MP Biomedical. The minimum levels of detection for ACTH and corticosterone were 5.7 pg/ml and 0.6 μg/dl, respectively. Intra-and interassay variability was less than 10%.

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Wild-type and OX2R knockout mice 30 mg/kg Oral administration

[1]. Discovery of 5''-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2':5',3''-terpyridine-3'-carboxamide (MK-1064): a selective orexin 2 receptor antagonist (2-SORA) for the treatment of insomnia. ChemMedChem. 2014 Feb;9(2):311-22.

[2]. Orexin 2 Receptor Antagonism is Sufficient to Promote NREM and REM Sleep from Mouse to Man. Sci Rep. 2016 Jun 3;6:27147.

[3]. Orexin 2 receptor regulation of the hypothalamic-pituitary-adrenal (HPA) response to acute and repeated stress. Neuroscience. 2017 Apr 21;348:313-323.

MK-1064 is under investigation in clinical trial NCT02549014 (A Single Dose Study of the Safety, Pharmacokinetics and Pharmacodynamics of MK-1064 (MK-1064-001)).

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The field of small-molecule orexin antagonist research has evolved rapidly in the last 15 years from the discovery of the orexin peptides to clinical proof-of-concept for the treatment of insomnia. Clinical programs have focused on the development of antagonists that reversibly block the action of endogenous peptides at both the orexin 1 and orexin 2 receptors (OX1R and OX2R), termed dual orexin receptor antagonists (DORAs), affording late-stage development candidates including Merck’s suvorexant (new drug application filed 2012). Full characterization of the pharmacology associated with antagonism of either OX1R or OX2R alone has been hampered by the dearth of suitable subtype-selective, orally bioavailable ligands. Herein, we report the development of a selective orexin 2 antagonist (2-SORA) series to afford a potent, orally bioavailable 2-SORA ligand. Several challenging medicinal chemistry issues were identified and overcome during the development of these 2,5-disubstituted nicotinamides, including reversible CYP inhibition, physiochemical properties, P-glycoprotein efflux and bioactivation. This article highlights structural modifications the team utilized to drive compound design, as well as in vivo characterization of our 2-SORA clinical candidate, 5′′-chloro-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2,2′:5′,3′′-terpyridine-3′-carboxamide (MK-1064), in mouse, rat, dog, and rhesus sleep models.[1]

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Orexin neuropeptides regulate sleep/wake through orexin receptors (OX1R, OX2R); OX2R is the predominant mediator of arousal promotion. The potential for single OX2R antagonism to effectively promote sleep has yet to be demonstrated in humans. MK-1064 is an OX2R-single antagonist. Preclinically, MK-1064 promotes sleep and increases both rapid eye movement (REM) and non-REM (NREM) sleep in rats at OX2R occupancies higher than the range observed for dual orexin receptor antagonists. Similar to dual antagonists, MK-1064 increases NREM and REM sleep in dogs without inducing cataplexy. Two Phase I studies in healthy human subjects evaluated safety, tolerability, pharmacokinetics and sleep-promoting effects of MK-1064, and demonstrated dose-dependent increases in subjective somnolence (via Karolinska Sleepiness Scale and Visual Analogue Scale measures) and sleep (via polysomnography), including increased REM and NREM sleep. Thus, selective OX2R antagonism is sufficient to promote REM and NREM sleep across species, similarly to that seen with dual orexin receptor antagonism.[2]

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Orexins are hypothalamic neuropeptides that have a documented role in mediating the acute stress response. However, their role in habituation to repeated stress, and the role of orexin receptors (OX1R and OX2R) in the stress response, has yet to be defined. Orexin neuronal activation and levels in the cerebrospinal fluid (CSF) were found to be stimulated with acute restraint, but were significantly reduced by day five of repeated restraint. As certain disease states such as panic disorder are associated with increased central orexin levels and failure to habituate to repeated stress, the effect of activating orexin signaling via Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) on the hypothalamic-pituitary-adrenal (HPA) response was evaluated after repeated restraint. While vehicle-treated rats displayed habituation of Adrenocorticotropic Hormone (ACTH) from day 1 to day 5 of restraint, stimulating orexins did not further increase ACTH beyond vehicle levels for either acute or repeated restraint. We delineated the roles of orexin receptors in acute and repeated stress using a selective OX2R antagonist (MK-1064). Pretreatment with MK-1064 reduced day 1 ACTH levels, but did not allow further habituation on day 5 compared with vehicle-treated rats, indicating that endogenous OX2R activity plays a role in acute stress, but not in habituation to repeated stress. However, in restrained rats with further stimulated orexins by DREADDs, MK-1064 decreased ACTH levels on day 5. Collectively, these results indicate that the OX2R plays a role in acute stress, and can prevent habituation to repeated stress under conditions of high orexin release.[3]

C24H20CLN5O3

461.91

461.125

C, 62.41; H, 4.36; Cl, 7.67; N, 15.16; O, 10.39

1207253-08-4

44633765

White to off-white solid powder

1.3±0.1 g/cm3

662.4±55.0 °C at 760 mmHg

354.4±31.5 °C

0.0±2.0 mmHg at 25°C

1.619

3.04

1

7

7

33

629

0

ClC1=C([H])N=C([H])C(=C1[H])C1C([H])=NC(C2=C([H])C([H])=C([H])C([H])=N2)=C(C(N([H])C([H])([H])C2C([H])=C([H])C(=C(N=2)OC([H])([H])[H])OC([H])([H])[H])=O)C=1[H]

CKTWQGHVNRYNCM-UHFFFAOYSA-N

InChI=1S/C24H20ClN5O3/c1-32-21-7-6-18(30-24(21)33-2)14-29-23(31)19-10-16(15-9-17(25)13-26-11-15)12-28-22(19)20-5-3-4-8-27-20/h3-13H,14H2,1-2H3,(H,29,31)

5-(5-chloropyridin-3-yl)-N-[(5,6-dimethoxypyridin-2-yl)methyl]-2-pyridin-2-ylpyridine-3-carboxamide

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.41 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)