MK-1064

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]

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。

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在重组人OX2R放射性配体结合实验中，MK-1064 可竞争性置换[125I]-食欲素A，亲和力高（Ki = 0.4 nM），而对人OX1R的结合力弱（Ki = 44 nM），显示出高OX2R选择性[1]
- 在稳定表达人OX2R的CHO细胞中，MK-1064 剂量依赖性抑制食欲素A诱导的钙内流（IC50 = 1.8 nM）；相比之下，对人OX1R介导的钙内流IC50为56 nM，证实其对OX2R的功能选择性[1]
- MK-1064（10 μM）对72种其他受体、离子通道或酶（如GABAA受体、NMDA受体、CYP450亚型）无显著结合活性，表明其脱靶选择性高[1]
- 全细胞膜片钳实验显示，在新生C57BL/6小鼠原代下丘脑神经元中，MK-1064（1-10 nM）可抑制食欲素A诱导的动作电位发放，抑制率为60-80%，且不影响神经元自发活性[2]

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。

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在植入EEG/EMG电极的C57BL/6小鼠中，口服MK-1064（3 mg/kg、10 mg/kg、30 mg/kg）可剂量依赖性增加24小时总睡眠时间（TST），较溶媒组分别增加15%、32%和45%；NREM睡眠时间分别增加18%、35%和48%，REM睡眠时间分别增加12%、28%和38%[2]
- 在大鼠中，口服MK-1064（1 mg/kg、3 mg/kg、10 mg/kg）可将入睡潜伏期从18 ± 3分钟缩短至12 ± 2、8 ± 1和6 ± 1分钟，并延长睡眠持续时间20-50%，且不改变睡眠结构（NREM/REM比值）[2]
- 在比格犬中，口服MK-1064（1 mg/kg、3 mg/kg）较溶媒组增加总睡眠时间25%和40%，NREM睡眠片段化（觉醒次数）分别减少30%和45%[2]
- 在健康志愿者（n=24）中，单次口服MK-1064（10 mg、20 mg、40 mg）可剂量依赖性增加夜间总睡眠时间（较安慰剂组分别增加10%、18%、25%），缩短入睡潜伏期（分别减少15%、28%、35%），延长NREM睡眠时间（分别增加12%、20%、27%）；REM睡眠时间分别增加8%、15%、22%，且无日间嗜睡或认知功能损伤[2]
- 在急性束缚应激（30分钟）大鼠中，口服MK-1064（3 mg/kg、10 mg/kg）可使血清皮质酮水平较溶媒组分别降低30%和55%，下丘脑CRF mRNA表达分别降低25%和40%[3]
- 在重复束缚应激（7天）大鼠中，口服MK-1064（10 mg/kg/天）可改善HPA轴反应的习惯化缺陷，血清ACTH水平较应激溶媒组降低45%[3]

人OX2R/OX1R放射性配体结合实验：将表达人OX2R或OX1R的重组细胞膜悬浮于结合缓冲液中，与[125I]-食欲素A（放射性配体）和系列稀释的MK-1064 混合，室温孵育60分钟。通过预浸泡结合缓冲液的玻璃纤维滤膜真空过滤分离结合态与游离态配体，使用γ计数器检测滤膜放射性，通过竞争结合曲线的非线性回归分析计算Ki值[1]
- 啮齿类动物OX2R结合实验：制备表达小鼠或大鼠OX2R的CHO细胞膜，采用上述相同方法进行结合实验（以[125I]-食欲素A为放射性配体），测定对啮齿类OX2R的Ki值以评估种属交叉反应性[1]
- 钙内流功能实验：将稳定表达人OX2R的CHO细胞接种到96孔板中，37°C负载钙敏感性荧光染料Fluo-4 AM 30分钟。加入系列稀释的MK-1064 预孵育15分钟，随后加入食欲素A（EC80浓度）诱导钙内流，酶标仪连续300秒监测荧光强度变化，基于食欲素A诱导的荧光反应抑制率计算IC50值[1]

原代下丘脑神经元电生理实验：从新生C57BL/6小鼠中分离下丘脑组织，解离为单细胞后接种到多聚L-赖氨酸包被的盖玻片上。培养7天后，神经元用人工脑脊液（ACSF）灌流，进行全细胞膜片钳记录。向灌流液中加入MK-1064（1-10 nM），随后加入食欲素A（100 nM）诱导动作电位发放，记录动作电位频率和振幅以评估MK-1064 对食欲素介导的神经元激活的抑制作用[2]
- OX2R选择性细胞实验：将表达人OX1R、GABAA受体、NMDA受体或多种离子通道的CHO细胞接种到96孔板中。用MK-1064（10 μM）和相应激动剂（如OX1R用食欲素A，GABAA受体用GABA）处理细胞，测定功能反应（钙内流、电流变化）以证实脱靶选择性[1]

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

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;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。