MK-1064

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

体外活性：MK-1064是一种新型、强效、选择性、口服生物可利用的Orexin OX2受体拮抗剂，具有用于治疗失眠的潜力。食欲素神经肽通过食欲素受体（OX1R、OX2R）调节睡眠/觉醒； OX2R 是唤醒促进的主要调节因子。 MK-1064 是一种 OX2R 单一拮抗剂。临床前，MK-1064 可以促进睡眠，并增加大鼠的快速眼动 (REM) 和非快速眼动 (NREM) 睡眠，OX2R 占用率高于双食欲素受体拮抗剂观察到的范围。

与双重拮抗剂类似，MK-1064 可以增加狗的 NREM 和 REM 睡眠，而不诱发猝倒。两项针对健康人类受试者的 I 期研究评估了 MK-1064 的安全性、耐受性、药代动力学和睡眠促进作用，并证明主观嗜睡（通过卡罗林斯卡嗜睡量表和视觉模拟量表测量）和睡眠（通过多导睡眠图）呈剂量依赖性增加，包括增加 REM 和 NREM 睡眠。因此，选择性 OX2R 拮抗作用足以促进跨物种的 REM 和 NREM 睡眠，类似于双重食欲素受体拮抗作用。 MK-1064 可促进睡眠并增加大鼠的快速眼动 (REM) 和非快速眼动 (NREM) 睡眠，OX2R 占用率高于双食欲素受体拮抗剂观察到的范围。 MK-1064 可以增加狗的 NREM 和 REM 睡眠，而不诱发猝倒。动物给药参考值为30mg/kg。

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

99.1

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.41 mM) (饱和度未知) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80+，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。