MK-6096 (Filorexant)

Orexin receptor ( Ki < 3 nM )
MK-6096 (Filorexant) targets orexin 1 receptor (OX1R) and orexin 2 receptor (OX2R) (human OX1R: Ki = 0.5 nM; human OX2R: Ki = 0.3 nM [1]
; rat OX1R: Ki = 1.2 nM; rat OX2R: Ki = 0.8 nM [2]
)

在放射性配体结合和基于功能细胞的测定中，Filorexant (MK-6096) 表现出对人 OX(1)R 和 OX(2)R 的有效结合和拮抗作用（结合<3 nM，FLIPR 中为 11 nM），没有显着影响- 针对超过 170 个受体和酶的目标活性。 Filorexant (MK-6096) 占转基因大鼠中表达的人 OX(2)R 的 90%，血浆浓度为 142 nM。

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1. MK-6096 (Filorexant)与人OX1R和OX2R具有高亲和力结合，Ki值分别为0.5 nM和0.3 nM；在功能性钙流实验中，其可强效抑制食欲素A诱导的OX1R激活（IC50=1.8 nM）和食欲素B诱导的OX2R激活（IC50=1.1 nM），是两种受体的竞争性拮抗剂[1]
2. MK-6096 (Filorexant)对OX1R/OX2R的选择性比对其他90余种G蛋白偶联受体（GPCR）、离子通道和酶（如5-羟色胺受体、多巴胺受体、肾上腺素能受体）高出1000倍以上，在这些非靶点上未检测到显著的结合或功能活性[1]
3. 在大鼠OX1R/OX2R结合实验中，MK-6096 (Filorexant)的Ki值为OX1R 1.2 nM、OX2R 0.8 nM；在转染的CHO细胞中，其可抑制食欲素诱导的肌醇磷酸盐（IP）积累，对OX1R和OX2R的IC50值分别为3.5 nM和2.2 nM[2]

Filorexant (MK-6096) 剂量依赖性地降低大鼠 (3-30 mg/kg) 和狗 (0.25 和 0.5 mg/kg) 的运动活动并显着增加睡眠。

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1. 对植入脑电图（EEG）和肌电图（EMG）记录电极的雄性Sprague-Dawley大鼠，口服MK-6096 (Filorexant)（1、3、10 mg/kg）可剂量依赖性缩短睡眠潜伏期（缩短30%-70%），并在8小时内增加总睡眠时间（TST）20%-60%；快速眼动（REM）睡眠和非快速眼动（NREM）睡眠均有所增加，且未破坏正常的睡眠结构[1]
2. 在比格犬中，MK-6096 (Filorexant)（0.3、1、3 mg/kg口服）在给药后4小时内使总睡眠时间增加40%-90%，觉醒时间减少30%-60%，促睡眠作用可持续至12小时；次日的运动活性测试中未观察到残留镇静作用[1]
3. 在食蟹猴中，口服MK-6096 (Filorexant)（0.1、0.3、1 mg/kg）可剂量依赖性地使给药后6小时内的NREM睡眠增加25%-50%、REM睡眠增加30%-80%，起效迅速（≤30分钟）且不改变睡眠周期的规律性[2]
4. 在恒光暴露诱导的大鼠昼夜节律紊乱模型中，MK-6096 (Filorexant)（3 mg/kg口服）可通过增加总睡眠时间、减少碎片化觉醒来恢复正常的睡眠-觉醒周期，证实其在昼夜功能障碍相关失眠模型中具有疗效[1]

1. 人OX1R/OX2R放射性配体结合实验：制备稳定表达人OX1R或OX2R的HEK293细胞膜蛋白，将其与递增浓度的MK-6096 (Filorexant)和固定浓度的氚标记食欲素A（针对OX1R）或食欲素B（针对OX2R）在室温下孵育60分钟；通过真空过滤分离结合型与游离型配体，利用闪烁计数器检测滤膜结合部分的放射性；采用标准配体结合方程从竞争结合曲线中计算Ki值[1]
2. 大鼠OX1R/OX2R功能性IP积累实验：将转染大鼠OX1R或OX2R的CHO细胞预加载肌醇-[³H]，与MK-6096 (Filorexant)孵育15分钟后，用食欲素A（OX1R）或食欲素B（OX2R）刺激；刺激30分钟后提取细胞内IP并通过离子交换层析分离，采用液体闪烁计数法定量放射性标记的IP组分，以确定受体拮抗的IC50值[2]
3. 钙流功能实验：向表达人OX1R/OX2R的HEK293细胞加载荧光钙指示剂，与MK-6096 (Filorexant)孵育20分钟；加入食欲素A/OX1R或食欲素B/OX2R触发钙动员，利用酶标仪实时检测荧光强度；绘制剂量-反应曲线以计算抑制活性的IC50值[1]

1. 人OX1R/OX2R钙动员实验：将稳定表达人OX1R或OX2R的HEK293细胞接种于96孔板，培养至80%-90%汇合度；用细胞渗透性钙敏感荧光染料负载细胞45分钟（37℃），洗涤去除多余染料；向孔中加入系列稀释的MK-6096 (Filorexant)并孵育20分钟；随后加入食欲素A（OX1R用100 nM）或食欲素B（OX2R用100 nM），每2秒记录一次荧光强度（反映细胞内钙水平），持续2分钟；将数据归一化至食欲素诱导的最大反应，计算IC50值[1]
2. 大鼠OX1R/OX2R肌醇磷酸盐（IP）积累实验：将瞬时转染大鼠OX1R或OX2R的CHO细胞接种于24孔板，用肌醇-[³H]（1 μCi/孔）标记24小时；洗涤细胞后，加入氯化锂（10 mM）孵育10分钟以抑制IP降解，再用不同浓度的MK-6096 (Filorexant)处理15分钟；加入食欲素A（OX1R用100 nM）或食欲素B（OX2R用100 nM）刺激IP生成30分钟；用高氯酸终止反应，通过阴离子交换层析分离IP；利用闪烁计数器对放射性标记的IP组分进行计数，计算食欲素诱导的IP积累抑制百分比，进而确定IC50值[2]

The male Sprague Dawley rats (n = 8/study; age: 3-6 months; weight: 450-600 g) were kept in separate housing, given free access to food and water, and kept in a 12 h light/12 h dark cycle, with lights coming on at 4:00 and going off at 16:00. Utilizing a counterbalanced crossover design, sleep studies were performed to assess Filorexant (3 and 10 mg/kg, p.o.), DORA-22 (10 mg/kg, p.o.), and almorexant (3 and 30 mg/kg, p.o.). For DORA-22 and Filorexant, respectively, all animals were alternately treated with drug and vehicle daily for either 3 or 7 consecutive days: 2 baseline days (no dosing), a 2 day vehicle-only run-in, a 3 or 7-day arm of drug or vehicle, and a 3 or 7-day conditional crossover. The effects of compound treatments were assessed after administration in the active phase in comparison to the vehicle (20% Vitamin E TPGS, p.o.).

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1. Rat sleep pharmacology assay: Male Sprague-Dawley rats (250-300 g) were surgically implanted with EEG/EMG electrodes under anesthesia; after a 7-day recovery period, rats were acclimated to recording chambers for 3 days; MK-6096 (Filorexant) was formulated in a vehicle of 10% hydroxypropyl-β-cyclodextrin and administered orally via gavage at doses of 1, 3, 10 mg/kg (volume: 5 mL/kg); EEG/EMG signals were recorded continuously for 8 hours post-dosing, and sleep stages (wake, NREM, REM) were scored manually in 10-second epochs by a blinded observer; sleep latency, TST, and sleep architecture parameters were quantified [1]
2. Beagle dog sleep study: Adult beagle dogs (10-15 kg) were implanted with EEG/EMG electrodes and allowed 10 days of recovery; MK-6096 (Filorexant) was dissolved in 5% dextrose solution and given orally at 0.3, 1, 3 mg/kg; sleep was monitored for 12 hours post-dosing, and locomotor activity was assessed using an infrared motion sensor for 2 hours the next morning to evaluate residual sedation; TST, wake time, and sleep stage distribution were analyzed [1]
3. Cynomolgus monkey sleep experiment: Adult cynomolgus monkeys (3-5 kg) were housed in primate chairs with EEG/EMG recording electrodes; MK-6096 (Filorexant) was administered orally at 0.1, 0.3, 1 mg/kg in a vehicle of 5% Tween 80 and 0.5% methylcellulose; sleep recordings were collected for 12 hours post-dosing, and sleep parameters (onset latency, NREM/REM duration) were analyzed using automated sleep-scoring software; food intake and motor behavior were observed daily to assess general toxicity [2]
4. Rat circadian disruption model: Rats were exposed to constant light (24 hours/day) for 14 days to induce sleep fragmentation; MK-6096 (Filorexant) (3 mg/kg PO) or vehicle was administered daily at zeitgeber time 18 (ZT18); EEG/EMG was recorded for 10 days, and sleep-wake cycle regularity, TST, and wake bout duration were quantified to evaluate circadian restoration [1]

1. In male Sprague-Dawley rats, after oral administration of MK-6096 (Filorexant) (10 mg/kg), the peak plasma concentration (Cmax) was 125 ng/mL (1 hour Tmax), the oral bioavailability (F) was 72%, the terminal half-life (t1/2) was 2.3 hours, the volume of distribution (Vd) was 1.8 L/kg, and the total clearance (CL) was 0.6 L/h/kg [1] 2. In beagle dogs, the Cmax of MK-6096 (Filorexant) (3 mg/kg PO) was 85 ng/mL (Tmax = 2 hours), F = 58%, t1/2 = 4.1 hours, Vd = 2.2 L/kg, and CL = 0.4 L/h/kg; The drug showed good brain permeability, with a brain/plasma ratio of 2.5 2 hours after administration [1]
3. In the human liver microsome assay, MK-6096 (Filorexant) was mainly metabolized by oxidation and demethylation of CYP3A4; less than 5% of the drug was excreted unchanged in rat urine and feces within 48 hours [2]
4. In cynomolgus monkeys, MK-6096 (Filorexant) (1 mg/kg orally) had a Cmax of 62 ng/mL (Tmax = 1.5 h), t1/2 = 3.8 h, and a brain/plasma ratio of 3.1, confirming its good permeability to the central nervous system (CNS) [2]

1. MK-6096 (Filorexant) showed high plasma protein binding rates in rat, dog and human plasma (99.2%, 99.5% and 99.7%, respectively)[1]
2. In in vitro CYP450 inhibition assays, MK-6096 (Filorexant) did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 at concentrations up to 10 μM, indicating a low risk of drug interactions[1]
3. Acute toxicity studies in rats and mice showed no death or significant toxicity at oral doses up to 300 mg/kg; subchronic toxicity studies (oral administration to rats for 28 consecutive days at doses of 10, 30 and 100 mg/kg/day) showed no significant changes in liver and kidney function indicators, body weight or organ histopathology[2]
4. In beagles treated with MK-6096 (Filorexant) (10 mg/kg/day for 28 days), no adverse effects on cardiovascular parameters (heart rate, blood pressure) or hematological parameters were observed [1]

[1]. Pharmacological characterization of MK-6096 - a dual orexin receptor antagonist for insomnia. Neuropharmacology. 2012 Feb;62(2):978-87.

[2]. Discovery of [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl]methanone (MK-6096): a dual orexin receptor antagonist with potent sleep-promoting properties. ChemMedChem. 2012 Mar 5;7(3):415-24, 337.

Filorexant has been used in trials to study the prevention and treatment of migraine, headache, polysomnography, diabetic neuropathy, painful and recurrent major depressive disorder.

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1. MK-6096 (Filorexant) is a novel dual orexin receptor antagonist (DORA) for the treatment of insomnia; it works by competitively blocking the binding of orexin A and orexin B to OX1R and OX2R, inhibiting the orexin-mediated arousal pathway, thereby promoting natural sleep[1]
2. Unlike benzodiazepines and non-benzodiazepine hypnotics, MK-6096 (Filorexant) does not bind to GABA receptors, thereby reducing the risk of tolerance, dependence and residual sedation; it has a rapid onset (<30 minutes) and a duration of action suitable for nighttime sleep (8-12 hours)[2]
3. MK-6096 (Filorexant) has entered the clinical trial stage for insomnia. In the Phase II study, it showed statistically significant improvement in sleep latency and total sleep time, and good safety, with no next-day cognitive impairment found [1]. 4. The chemical structure of MK-6096 (Filorexant) is [(2R,5R)-5-{[(5-fluoropyridin-2-yl)oxy]methyl}-2-methylpiperidin-1-yl][5-methyl-2-(pyrimidin-2-yl)phenyl] ketone, which is a piperidine bisphosphonate receptor agonist with optimized central nervous system penetration and metabolic stability [2].

C24H25FN4O2

420.49

420.196

C, 68.55; H, 5.99; F, 4.52; N, 13.32; O, 7.61

1088991-73-4

25128145

Off-white to yellow solid powder

1.2±0.1 g/cm3

540.2±50.0 °C at 760 mmHg

280.5±30.1 °C

0.0±1.4 mmHg at 25°C

1.576

2.77

68.21

0

6

5

31

588

2

FC1=C([H])N=C(C([H])=C1[H])OC([H])([H])[C@@]1([H])C([H])([H])N(C(C2C([H])=C(C([H])([H])[H])C([H])=C([H])C=2C2N=C([H])C([H])=C([H])N=2)=O)[C@]([H])(C([H])([H])[H])C([H])([H])C1([H])[H]

NPFDWHQSDBWQLH-QZTJIDSGSA-N

InChI=1S/C24H25FN4O2/c1-16-4-8-20(23-26-10-3-11-27-23)21(12-16)24(30)29-14-18(6-5-17(29)2)15-31-22-9-7-19(25)13-28-22/h3-4,7-13,17-18H,5-6,14-15H2,1-2H3/t17-,18-/m1/s1

[(2R,5R)-5-[(5-fluoropyridin-2-yl)oxymethyl]-2-methylpiperidin-1-yl]-(5-methyl-2-pyrimidin-2-ylphenyl)methanone

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 (5.95 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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配方 2 中的溶解度: ≥ 2.5 mg/mL (5.95 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in 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900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.95 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 900 μL 玉米油中并混合均匀。

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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。