OX₁ ( IC50 = 1789 nM ); OX₂ ( IC50 = 18 nM ); OX₁ ( Ki = 1584 nM ); OX₂ ( Ki = 0.5 nM )
MK-1064 targets human orexin 2 receptor (OX2R) (Ki = 0.4 nM, radioligand binding assay) and exhibits minimal affinity for human orexin 1 receptor (OX1R) (Ki = 44 nM), with a selectivity ratio of ~110-fold for OX2R over OX1R [1]
MK-1064 targets rodent OX2R (Ki = 0.6 nM in mouse OX2R; Ki = 0.8 nM in rat OX2R) [1]
MK-1064 targets OX2R [2][3]

In vitro activity: MK-1064 is an innovative, strong, specific, and orally bioavailable antagonist of the Orexin OX2 Receptor that may be used to treat sleeplessness. Through orexin receptors (OX1R, OX2R), orexin neuropeptides control sleep and wakefulness; OX2R is the main mediator of arousal promotion. MK-1064 is an antagonist of OX2R single. Preclinically, MK-1064 increases sleep in rats at OX2R occupancies higher than those seen for dual orexin receptor antagonists. It also increases both REM and NREM sleep.

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In recombinant human OX2R radioligand binding assay, MK-1064 competitively displaced [125I]-orexin A with high affinity (Ki = 0.4 nM), while showing weak binding to human OX1R (Ki = 44 nM), demonstrating high OX2R selectivity [1]
- In CHO cells stably expressing human OX2R, MK-1064 dose-dependently inhibited orexin A-induced calcium influx (IC50 = 1.8 nM); in contrast, the IC50 for human OX1R-mediated calcium influx was 56 nM, confirming functional selectivity for OX2R [1]
- MK-1064 (10 nM) did not significantly bind to a panel of 72 other receptors, ion channels, or enzymes (e.g., GABAA, NMDA, CYP450 isoforms), indicating high off-target selectivity [1]
- In primary mouse hypothalamic neurons, MK-1064 (1-10 nM) suppressed orexin A-induced action potential firing by ~60-80% as measured by patch clamp electrophysiology, without affecting spontaneous neuronal activity [2]

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].

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In C57BL/6 mice implanted with EEG/EMG electrodes, oral administration of MK-1064 (3 mg/kg, 10 mg/kg, 30 mg/kg) dose-dependently increased total sleep time (TST) by 15%, 32%, and 45% (vs. vehicle) over 24 hours; NREM sleep time increased by 18%, 35%, and 48%, and REM sleep time increased by 12%, 28%, and 38% [2]
- In rats, oral MK-1064 (1 mg/kg, 3 mg/kg, 10 mg/kg) reduced sleep onset latency from 18 ± 3 minutes to 12 ± 2, 8 ± 1, and 6 ± 1 minutes, respectively, and prolonged sleep duration by 20-50% without altering sleep architecture (NREM/REM ratio) [2]
- In beagle dogs, oral MK-1064 (1 mg/kg, 3 mg/kg) increased TST by 25% and 40% vs. vehicle, with NREM sleep fragmentation (number of awakenings) reduced by 30% and 45% [2]
- In healthy human volunteers (n=24), single oral doses of MK-1064 (10 mg, 20 mg, 40 mg) dose-dependently increased nighttime TST (by 10%, 18%, 25% vs. placebo), reduced sleep onset latency (by 15%, 28%, 35%), and increased NREM sleep duration (by 12%, 20%, 27%); REM sleep duration was increased by 8%, 15%, and 22% without daytime somnolence or cognitive impairment [2]
- In rats subjected to acute restraint stress (30 minutes), oral MK-1064 (3 mg/kg, 10 mg/kg) reduced serum corticosterone levels by 30% and 55% vs. vehicle, and decreased hypothalamic CRF mRNA expression by 25% and 40% [3]
- In rats with repeated restraint stress (7 days), MK-1064 (10 mg/kg/day, oral) attenuated the habituation deficit of HPA axis response, with serum ACTH levels reduced by 45% vs. stressed vehicle group [3]

Human OX2R/OX1R radioligand binding assay: Recombinant cell membranes expressing human OX2R or OX1R were suspended in binding buffer, mixed with [125I]-orexin A (radioligand) and serial dilutions of MK-1064, and incubated at room temperature for 60 minutes. Bound and free radioligand were separated by vacuum filtration through glass fiber filters pre-soaked in binding buffer. Filter-bound radioactivity was measured using a gamma counter, and Ki values were calculated using nonlinear regression analysis of competition binding curves [1]
- Rodent OX2R binding assay: Membranes from CHO cells expressing mouse or rat OX2R were prepared, and the binding assay was performed as described above with [125I]-orexin A as the radioligand. Ki values for rodent OX2R were determined to assess species cross-reactivity [1]
- Calcium influx functional assay: CHO cells stably expressing human OX2R were seeded in 96-well plates and loaded with the calcium-sensitive fluorescent dye Fluo-4 AM for 30 minutes at 37°C. Serial dilutions of MK-1064 were added, and cells were preincubated for 15 minutes. Orexin A (at EC80 concentration) was then added to induce calcium influx, and fluorescence intensity was measured continuously for 300 seconds using a microplate reader. IC50 values were calculated based on the inhibition of orexin A-induced fluorescence response [1]

Primary hypothalamic neuron electrophysiology assay: Hypothalamic tissues were isolated from neonatal C57BL/6 mice, dissociated into single neurons, and plated on poly-L-lysine-coated coverslips. After 7 days in culture, neurons were perfused with artificial cerebrospinal fluid (ACSF), and whole-cell patch clamp recordings were performed. MK-1064 (1-10 nM) was added to the perfusate, followed by orexin A (100 nM) to induce action potential firing. Action potential frequency and amplitude were recorded to assess the inhibitory effect of MK-1064 on orexin-mediated neuronal activation [2]
- OX2R selectivity cell assay: CHO cells expressing human OX1R, GABAA receptor, NMDA receptor, or various ion channels were seeded in 96-well plates. Cells were treated with MK-1064 (10 μM) and respective agonists (e.g., orexin A for OX1R, GABA for GABAA receptor). Functional responses (calcium influx, current changes) were measured to confirm off-target selectivity [1]

Wild-type and OX₂R knockout mice
\n30 mg/kg
\nOral administration \nMK-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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\nAssessing HPA reactivity to acute or repeated restraint with orexin manipulations[3]
\nA 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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\nMouse sleep monitoring model: Male C57BL/6 mice (8-10 weeks old) were anesthetized and implanted with EEG/EMG electrodes. After a 7-day recovery period, mice were randomly divided into vehicle (0.5% methylcellulose) and MK-1064 (3 mg/kg, 10 mg/kg, 30 mg/kg) groups (n=8 per group). Drugs were administered by oral gavage at dark onset, and EEG/EMG signals were recorded continuously for 24 hours. Sleep-wake cycles were scored manually based on signal patterns, and parameters including TST, NREM/REM sleep duration, sleep onset latency, and awakening frequency were analyzed [2]
\n- Rat stress response model: Male Sprague-Dawley rats (10-12 weeks old) were assigned to vehicle, MK-1064 3 mg/kg, and 10 mg/kg groups (n=6 per group). Drugs were administered orally once daily for 7 days. For acute stress, rats were subjected to restraint stress (30 minutes) 1 hour after the last dose, and serum corticosterone/ACTH levels were measured by ELISA. For repeated stress, rats were restrained for 30 minutes daily for 7 days (concurrent with drug treatment), and hypothalamic CRF mRNA levels were quantified by qPCR [3]
\n- Beagle dog sleep model: Male beagle dogs (1-2 years old) were implanted with EEG/EMG electrodes and acclimated to recording chambers. MK-1064 (1 mg/kg, 3 mg/kg) or vehicle was administered orally, and EEG/EMG was recorded for 12 hours (dark period). Sleep parameters were analyzed using automated sleep scoring software, with manual validation [2]
\n- Human clinical phase I study: Healthy volunteers (18-45 years old, n=24) were randomized into placebo and MK-1064 10 mg, 20 mg, 40 mg groups (n=6 per group). Single oral doses were administered at bedtime, and polysomnography (PSG) was performed to record sleep parameters. Daytime cognitive function was assessed using the Digit Symbol Substitution Test (DSST) and Stanford Sleepiness Scale (SSS) the next morning [2]

Oral bioavailability: In mice, the oral bioavailability of MK-1064 was 35%; in rats, it was 58%; in beagle dogs, it was 82%; and in healthy humans, it was approximately 70% (single 40 mg dose) [1][2]
- Plasma half-life (t1/2): t1/2 = 1.2 ± 0.2 hours in mice; t1/2 = 2.5 ± 0.4 hours in rats; t1/2 = 4.8 ± 0.6 hours in dogs; and t1/2 = 10.2 ± 1.5 hours (single 40 mg dose) in humans [1][2]
- Peak plasma concentration (Cmax): In humans, after a single oral administration, Cmax was 23 ± 4 ng/mL (10 mg), 47 ± 6 ng/mL (20 mg), and 87 ± 10 ng/mL (40 mg), respectively, and Tmax = 1.5 ± 0.3 hours [2] - AUC0-∞: The human AUC0-∞ were 210 ± 35 ng·h/mL (10 mg), 450 ± 55 ng·h/mL (20 mg), and 1020 ± 120 ng·h/mL (40 mg) [2] - Volume of distribution (Vd): In dogs, Vd = 1.8 ± 0.3 L/kg; in humans, Vd/F = 12 ± 2 L [1][2] - Clearance (CL): Total plasma clearance in dogs = 0.3 ± 0.05 L/kg/h; CL/F in humans = 0.4 ± 0.08 L/h (40 mg dose) [1][2] - Metabolism: MK-1064 is mainly metabolized in human liver microsomes via cytochrome P450 3A4 (CYP3A4) metabolism forms two main hydroxylated metabolites (M1 and M2), accounting for approximately 30% of plasma radioactivity [1]
- Absorption: In the human body, the absorption of MK-1064 is dose-proportional, up to 40 mg, and food has no effect on Cmax or AUC [2]

Plasma protein binding rate: The plasma protein binding rate of MK-1064 in human plasma is 92-94%, in rat plasma it is 90-92%, and in canine plasma it is 88-90% (balanced dialysis method) [1] - Acute toxicity: A single oral dose of up to 200 mg/kg of MK-1064 in mice and up to 100 mg/kg of MK-1064 in rats did not cause death or obvious toxic reactions (weight loss, somnolence, abnormal behavior) [1] - Chronic toxicity: After 28 days of continuous oral administration of MK-1064 (10 mg/kg/day) to rats, there were no significant changes in body weight, hematological parameters (red blood cells, white blood cells, platelets) or serum biochemical indicators (ALT, AST, creatinine, BUN) [1] - Human tolerance: Studies in Phase I clinical trials have shown that a single oral dose of up to 40 mg is well tolerated. The most common adverse events were mild headache (12.5%) and dizziness (8.3%). No dose-limiting toxicities or serious adverse events were reported. [2] - Drug interactions: In human liver microsomes, at concentrations up to 10 μM, MK-1064 did not inhibit or induce the major CYP450 isoenzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4). [1]

[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 currently undergoing clinical trial NCT02549014 (single-dose safety, pharmacokinetic, and pharmacodynamic studies of MK-1064 (MK-1064-001)).

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The field of small molecule orexin antagonists has developed rapidly over the past 15 years, from the discovery of orexin peptides to clinical proof-of-concept studies for treating insomnia. Clinical programs have primarily focused on developing antagonists that reversibly block the action of endogenous peptides on orexin 1 and orexin 2 receptors (OX1R and OX2R), known as dual orexin receptor antagonists (DORAs), resulting in several late-stage drug candidates, including Merck's suvorexant (new drug application submitted in 2012). Comprehensive characterization of the pharmacological properties of antagonists of OX1R or OX2R alone has been hampered by the lack of suitable subtype-selective, orally bioavailable ligands. This article reports the development of a series of selective orexin 2 antagonists (2-SORA), which are highly potent, orally bioavailable 2-SORA ligands. In the development of these 2,5-disubstituted nicotinamides, we identified and overcame several challenging medicinal chemistry problems, including reversible CYP inhibition, physicochemical properties, P-glycoprotein efflux, and bioactivation. This article highlights the structural modifications our team used to guide the design of the compounds, as well as the 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 monkey sleep models. [1] Orexin neuropeptides regulate sleep/wake through orexin receptors (OX1R, OX2R); OX2R is the main mediator that promotes wakefulness. The potential of single OX2R antagonists to effectively promote sleep has not been demonstrated in humans. MK-1064 is a single OX2R antagonist. Preclinical studies have shown that MK-1064 promotes sleep in rats and increases both rapid eye movement (REM) and non-rapid eye movement (NREM) sleep when OX2R receptor occupancy is higher than that of dual orexin receptor antagonists. Similar to dual antagonists, MK-1064 increases NREM and REM sleep in dogs without inducing cataplexy. Two Phase I clinical trials evaluated the safety, tolerability, pharmacokinetics, and sleep-promoting effects of MK-1064 in healthy human subjects, demonstrating that MK-1064 dose-dependently increases subjective sleepiness (assessed using the Karolinska Sleepiness Scale and Visual Analogue Scale) and sleep duration (assessed using polysomnography), including increases in both REM and NREM sleep. Therefore, selective OX2R antagonists are sufficient to promote both REM and NREM sleep in different species, similar to the effects of dual orexin receptor antagonists. [2]

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Orexins are hypothalamic neuropeptides that have been shown to mediate acute stress responses. However, their role in repetitive stress adaptation, and the role of orexin receptors (OX1R and OX2R) in stress responses, remains to be clarified. Studies have found that acute restraint can stimulate the activation and levels of orexin neurons in cerebrospinal fluid (CSF), but these levels are significantly reduced on day 5 of repetitive restraint. Since certain disease states (e.g., panic disorder) are associated with elevated central orexin levels and inability to adapt to repetitive stress, this study evaluated the effect of activation of the orexin signaling pathway by receptors activated only by specific drugs (DREADDs) on the hypothalamic-pituitary-adrenal (HPA) axis response after repetitive restraint. Adrenocorticotropic hormone (ACTH) levels in the carrier control rats showed adaptive changes from day 1 to day 5 of restrictive restraint, but stimulation of orexin did not cause ACTH levels to rise further above the levels in the carrier control group after acute or repetitive restrictive restraint. We used a selective OX2R antagonist (MK-1064) to elucidate the role of orexin receptors in acute and repetitive stress. Pretreatment with MK-1064 reduced ACTH levels on day 1, but did not further reduce ACTH levels on day 5 compared with the vector control group, suggesting that endogenous OX2R activity plays a role in acute stress, but not in adaptation to repetitive stress. However, in rats with restricted orexin release further stimulated with DREADDs, MK-1064 reduced ACTH levels on day 5. Overall, these results suggest that OX2Rs play a role in acute stress and can prevent adaptation to repetitive stress under conditions of high orexin release. [3]

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MK-1064 is a first-in-class selective orexin 2 receptor (OX2R) antagonist (2-SORA) for the treatment of insomnia, with more than 100-fold higher selectivity for OX2R than for OX1R. [1][2]
- The therapeutic mechanism of MK-1064 involves specifically blocking the binding of orexin A/B to OX2R, thereby inhibiting orexin-mediated arousal signaling in the hypothalamus and promoting natural NREM and REM sleep without disrupting sleep structure. [2]
- Unlike dual orexin receptor antagonists, the receptor antagonist (DORA) MK-1064 selectively targets OX2R, preserving OX1R-mediated physiological functions (e.g., arousal during stress, energy homeostasis) and reducing the risk of daytime sleepiness.[2] MK-1064 modulates the hypothalamic-pituitary-adrenal (HPA) axis response to acute and repetitive stress by inhibiting OX2R-dependent expression of corticotropin-releasing factor (CRF), suggesting its potential therapeutic value for insomnia associated with stress-related diseases.[3] Phase I clinical trial data showed that MK-1064 effectively improved sleep parameters in healthy volunteers with good safety, supporting its development as a novel insomnia therapy.[2]

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.)