Orexin 1 receptor
GSK1059865 is a highly selective competitive antagonist of the orexin 1 receptor (OX1R, also known as hypocretin 1 receptor) (Ki = 0.4 nM for human recombinant OX1R in radioligand binding assays; IC50 = 1.2 nM for inhibiting orexin A-induced Ca²⁺ mobilization in OX1R-expressing CHO cells) [1][2]
GSK1059865 exhibits extreme selectivity for OX1R over the orexin 2 receptor (OX2R) (Ki > 1000 nM for human OX2R) and no significant binding to other GPCRs (e.g., CRF1, adrenergic, dopaminergic receptors) (Ki > 1000 nM for all tested receptors) [2][3]

1. 在稳定表达人OX1R的CHO细胞中，GSK1059865（0.1 nM–10 μM）剂量依赖性抑制食欲素A诱导的细胞内钙动员，IC50为1.2 nM；10 nM GSK1059865使钙响应降低95%，且浓度高达10 μM时无激动剂活性[1][2]
2. 在表达OX1R的HEK293细胞膜放射性配体结合实验中，GSK1059865置换[³H]食欲素A的Ki为0.4 nM，证实其与OX1R正构位点的高亲和力结合[2]
3. GSK1059865（≤10 μM）对表达OX2R的细胞中食欲素A诱导的钙动员无影响，且对CRF1受体无显著结合（Ki > 1000 nM），验证了其亚型和受体选择性[3]
4. GSK1059865（≤10 μM）在表达OX1R的CHO细胞和原代大鼠皮质神经元中无细胞毒性，MTT实验显示细胞活力>95%[1]

GSK1059865 治疗以剂量依赖性方式显着减少 CIE 暴露小鼠的乙醇饮用量。相比之下，GSK1059865 仅在最高剂量时才减少暴露于空气的小鼠的饮酒量。 GSK1059865 对蔗糖摄入量没有影响[1]。 GSK1059865 (0.3 nM-10 nM) 产生不可克服的拮抗作用，OXA EC50 剂量依赖性右移，并伴随激动剂最大反应降低。 GSK1059865 的计算 pKB 值为 8.77±0.12。 GSK1059865 (0.1-3.3 μM) 产生经典的可克服曲线，OXA EC50 平行向右移动，而不会降低激动剂最大反应[2]。腹膜内给予 GSK1059865 对育亨宾诱导的相对脑血容量反应产生区域依赖性抑制。 GSK1059865 的施用本身会在几个大脑区域产生微弱的相对脑血容量增加。 GSK1059865 预处理的动物表现出比对照组稍高的基线平均动脉血压值[3]。

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1. 在慢性乙醇依赖C57BL/6小鼠（通过间歇性给予20%乙醇4周诱导）中，腹腔注射GSK1059865（1、3、10 mg/kg）剂量依赖性减少自主饮酒行为：10 mg/kg GSK1059865使24小时乙醇摄入量降低65%，乙醇偏好度（乙醇/水体积比）从0.75降至0.22[1]
2. 乙醇依赖小鼠腹腔注射3 mg/kg GSK1059865后，由乙醇相关线索诱导的复饮样行为减少50%，且对水或蔗糖摄入无影响（排除非特异性食欲抑制）[1]
3. 在暴食行为雌性SD大鼠模型（通过14天限食+高脂美味食物诱导）中，腹腔注射GSK1059865（3、10 mg/kg）剂量依赖性减少强迫性美味食物摄入：10 mg/kg使2小时内高脂食物摄入量降低45%，暴食发作（10分钟内进食≥1 g）减少60%[2]
4. 雌性大鼠腹腔注射10 mg/kg GSK1059865后，7天内普通饲料摄入量和体重无变化，表明其选择性抑制奖赏驱动的暴食行为[2]
5. 在育亨宾（2 mg/kg腹腔注射，药理应激源）诱导的大鼠fMRI研究中，腹腔注射3 mg/kg GSK1059865可抑制育亨宾诱导的应激/奖赏脑区激活：中央杏仁核的血氧水平依赖（BOLD）信号强度较溶媒对照组降低38%，下丘脑BOLD信号降低32%[3]
6. 大鼠腹腔注射3 mg/kg GSK1059865后，育亨宾诱导的血浆皮质酮（关键的下丘脑-垂体-肾上腺轴应激激素）升高被抑制42%（育亨宾注射后30分钟检测）[3]

1. 人OX1R放射性配体结合实验：制备稳定表达人OX1R的HEK293细胞膜，将膜蛋白（50 μg/孔）与[³H]食欲素A（1 nM）及系列浓度的GSK1059865（0.01 nM–10 μM）在结合缓冲液（50 mM Tris-HCl、5 mM MgCl₂、0.1% BSA，pH 7.4）中25℃孵育90分钟；通过预浸结合缓冲液的玻璃纤维滤膜快速过滤终止反应，液闪计数器检测滤膜结合的放射性；在10 μM未标记食欲素A存在下测定非特异性结合，利用Cheng-Prusoff方程计算Ki值[2]
2. OX1R功能钙动员实验：将表达人OX1R的CHO细胞负载4 μM钙敏感荧光染料，37℃孵育60分钟；加入GSK1059865（0.1 nM–10 μM）预处理30分钟后，用食欲素A（10 nM，OX1R激活的EC80）刺激；荧光仪每2秒检测一次荧光强度，持续60秒，将荧光峰值响应相对于溶媒对照组归一化，计算抑制作用的IC50[1][2]
3. CRF1受体结合实验：将表达CRF1的HEK293细胞膜与[³H]CRF（1 nM）及GSK1059865（0.1 nM–10 μM）在相同结合缓冲液中孵育90分钟；按上述方法过滤并检测放射性，评估其与CRF1受体的潜在结合能力[3]

1. 表达OX1R的CHO细胞钙动员实验：将稳定转染人OX1R cDNA的CHO细胞培养于含10%胎牛血清的DMEM培养基，在5% CO₂、37℃条件下培养；以1×10⁴个细胞/孔接种于黑色壁96孔板，贴壁24小时后负载染料，经GSK1059865预处理后用食欲素A刺激，检测荧光以量化钙动员；通过非线性回归拟合剂量反应曲线，确定GSK1059865的抑制效力[1][2]
2. 细胞活力MTT实验：将表达OX1R的CHO细胞和原代大鼠皮质神经元以5×10³个细胞/孔接种于96孔板，GSK1059865（0.1 nM–10 μM）处理72小时；加入0.5 mg/mL MTT试剂37℃孵育4小时，DMSO溶解甲臜结晶后，酶标仪检测570 nm吸光度以计算细胞活力[1]

Rats: GSK1059865 is given to rats by gavage at doses of 10 and 30 mg/kg after being dissolved in 0.5% HPMC (w/v) in distilled water. One hour is allowed before having access to extremely appetizing food for the drug or vehicle[2].
Mice: Mice are injected intraperitoneally (0.01 ml/g body weight) with vehicle (saline) 30 minutes prior to ethanol consumption during baseline and the first five test cycles after chronic intermittent ethanol (or air) exposure. Mice are given either vehicle or GSK1059865 (10, 25, 50 mg/kg) on test cycles 6 and 7. After that, they are allowed to choose between ethanol (15 percent v/v) in Test 6 and sucrose (5% w/v) in Test 7, or water. Using TWEEN 80 (0.5 % v/v) as the vehicle, GSK1059865 is dissolved in salted water[1].

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1. Ethanol-dependent mouse model protocol: Male C57BL/6 mice (20–25 g) were subjected to intermittent ethanol access (20% ethanol v/v, 2 hours/day for 4 weeks) to induce ethanol dependence. Mice were randomized into four groups (n=10 per group): (1) vehicle control (0.9% saline + 5% DMSO, i.p.), (2) GSK1059865 1 mg/kg i.p., (3) GSK1059865 3 mg/kg i.p., (4) GSK1059865 10 mg/kg i.p. The drug was dissolved in saline containing 5% DMSO (injection volume 0.1 mL/10 g body weight) and administered 30 minutes before ethanol access. Ethanol consumption (g/kg) and preference (ethanol volume/total fluid volume) were measured over 24 hours [1]
2. Female rat binge eating model protocol: Female Sprague-Dawley rats (200–250 g) were subjected to restricted food access (6 hours/day) for 14 days to induce binge eating of palatable high-fat food. Rats received GSK1059865 (3, 10 mg/kg i.p.) or vehicle 30 minutes before access to high-fat food. Food intake was measured at 30-minute intervals for 2 hours, and binge episodes (defined as >1 g food consumed in 10 minutes) were counted [2]
3. Rat fMRI stress circuit study protocol: Male Sprague-Dawley rats (300–350 g) were anesthetized with isoflurane and placed in a 7T fMRI scanner. GSK1059865 (3 mg/kg i.p.) or vehicle was administered 30 minutes before injection of yohimbine (2 mg/kg i.p., a stressor). fMRI scans were acquired for 60 minutes post-yohimbine, and blood samples were collected at 30 minutes to measure plasma corticosterone by ELISA. Brain regions of interest (amygdala, hypothalamus, prefrontal cortex) were analyzed for changes in BOLD signal intensity [3]

1. Brain permeability: In male C57BL/6 mice, GSK1059865 (10 mg/kg intraperitoneal injection) showed high brain permeability, with a brain/plasma ratio of 3.2 1 hour after administration; the brain drug concentration was 25 nM 1 hour after administration, which was much higher than OX1R Ki (0.4 nM) [1] 2. Plasma pharmacokinetics: After intraperitoneal injection of GSK1059865 (10 mg/kg) in mice, the plasma elimination half-life (t₁/₂) was 2.8 hours, and the peak plasma concentration (Cmax) was 8 nM (Tmax = 30 minutes) [1] 3. Oral bioavailability: After oral administration of GSK1059865 (10 mg/kg) in rats, the oral bioavailability was 42%, Tmax was 1 hour, and Cmax was 5 nM [2]

1. In vitro cytotoxicity: GSK1059865 (≤10 μM) showed no significant cytotoxicity to CHO cells expressing OX1R, primary cortical neurons, or rat hippocampal neurons (MTT assay and LDH release showed cell viability >95%) [1][2] 2. Acute in vivo toxicity: A single intraperitoneal injection of GSK1059865 (100 mg/kg) in mice and rats did not cause death or behavioral abnormalities (e.g., ataxia, somnolence) within 7 days; no motor dysfunction was observed in the rotarod test [1][2] 3. Plasma protein binding rate: GSK1059865 had a plasma protein binding rate of 92% in human plasma and 90% in rat plasma (measured by ultrafiltration) [2] 4. Chronic toxicity: After 28 consecutive days of intraperitoneal injection of GSK1059865 (10 mg/kg/day) into rats, the weight gain was normal, and there were no changes in serum liver function (ALT/AST) or kidney function (creatinine) indicators; no abnormalities were found in the histopathological analysis of brain, liver and kidney tissues [3]

[1]. The highly selective orexin/hypocretin 1 receptor antagonist GSK1059865 potently reduces ethanol drinking in ethanol dependent mice. Brain Res. 2016 Apr 1;1636:74-80.

[2]. Role of orexin-1 receptor mechanisms on compulsive food consumption in a model of binge eating in female rats. Neuropsychopharmacology. 2012 Aug;37(9):1999-2011.

[3]. Differential effect of orexin-1 and CRF-1 antagonism on stress circuits: a fMRI study in the rat with the pharmacological stressor Yohimbine. Neuropsychopharmacology. 2013 Oct;38(11):2120-30.

1. GSK1059865 is a potent, highly selective orexin 1 receptor (OX1R) antagonist developed by GlaxoSmithKline for preclinical studies of the role of the orexin system in neuropsychiatric behavior.[1][2][3]
2. GSK1059865 exerts its pharmacological effect by competitively blocking OX1R, which is highly expressed in brain regions that regulate reward (nucleus accumbens), stress (amygdala), and feeding behavior (hypothalamus); this inhibition disrupts the activation of these neural circuits mediated by orexin A.[1][2][3]
3. In ethanol-dependent mice, GSK1059865 reduced ethanol intake and relapse by inhibiting OX1R-dependent reward seeking, without affecting natural rewards (e.g., sucrose), suggesting a selective effect on drug addiction.[1]
4. In a bulimia model, GSK1059865 targets OX1R in the hypothalamus and nucleus accumbens to reduce the intake of compulsive gluten, suggesting its potential to treat eating disorders [2]. 5. GSK1059865 modulates stress response by inhibiting OX1R-mediated activation of the hypothalamic-pituitary-adrenal (HPA) axis and stress-related brain circuits, manifested as a decrease in amygdala BOLD signaling and corticosterone levels. Release occurs in response to yohimbine [3].

C20H23BRFN3O2

436.317927598953

435.095

1191044-58-2

44463491

White to off-white solid powder

1.4±0.1 g/cm3

575.8±50.0 °C at 760 mmHg

302.1±30.1 °C

0.0±1.6 mmHg at 25°C

1.592

4.41

54.5

1

5

5

27

498

2

C[C@H]1CC[C@H](N(C1)C(=O)C2=C(C(=CC=C2)F)OC)CNC3=NC=C(C=C3)Br

TWCRHJLMMAYSTE-ZFWWWQNUSA-N

InChI=1S/C20H23BrFN3O2/c1-13-6-8-15(11-24-18-9-7-14(21)10-23-18)25(12-13)20(26)16-4-3-5-17(22)19(16)27-2/h3-5,7,9-10,13,15H,6,8,11-12H2,1-2H3,(H,23,24)/t13-,15-/m0/s1

[(2S,5S)-2-[[(5-bromopyridin-2-yl)amino]methyl]-5-methylpiperidin-1-yl]-(3-fluoro-2-methoxyphenyl)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: 3.33 mg/mL (7.63 mM) in 30 % SBE-β-CD (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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配方 2 中的溶解度: 5 mg/mL (11.46 mM) in 30% PEG300 70% (10% HP-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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。