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. In CHO cells stably expressing human OX1R, GSK1059865 (0.1 nM–10 μM) dose-dependently inhibits orexin A-induced intracellular Ca²⁺ mobilization with an IC50 of 1.2 nM; 10 nM GSK1059865 reduces the Ca²⁺ response by 95% and shows no agonist activity at concentrations up to 10 μM [1][2]
2. In radioligand binding assays using membranes from OX1R-expressing HEK293 cells, GSK1059865 displaces [³H]orexin A with a Ki of 0.4 nM, confirming high-affinity binding to the OX1R orthosteric site [2]
3. GSK1059865 (≤10 μM) has no effect on orexin A-induced Ca²⁺ mobilization in OX2R-expressing cells, and no significant binding to the CRF1 receptor (Ki > 1000 nM), verifying its subtype and receptor selectivity [3]
4. GSK1059865 (≤10 μM) shows no cytotoxicity in OX1R-expressing CHO cells or primary rat cortical neurons (cell viability >95% by MTT assay) [1]

GSK1059865 treatment dramatically reduces ethanol consumption in CIE-exposed mice in a dose-dependent manner. On the other hand, GSK1059865 only reduces drinking at the maximum dose when given to mice exposed to air. GSK1059865 has no effect on the consumption of sucrose[1]. Non-surmountable antagonism is produced by GSK1059865 (0.3 nM–10 nM), which also causes a dose-dependent rightward shift of the OXA EC50 and a concurrent reduction in the agonist maximal response. The calculated pKB value is 8.77±0.12 for GSK1059865. GSK1059865 (0.1-3.3 μM) generates a classical surmountable profile with a parallel rightward shift of the OXA EC50 without lowering the agonist maximal response[2]. The administration of GSK1059865 intraperitoneally results in a region-specific suppression of the relative cerebral blood volume response induced by yohimbine. In various brain regions, the administration of GSK1059865 alone results in a modest relative increase in cerebral blood volume. Animals pretreated with GSK1059865 have baseline mean arterial blood pressure values that are marginally higher than controls[3].

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1. In C57BL/6 mice with chronic ethanol dependence (induced by intermittent 20% ethanol access for 4 weeks), intraperitoneal (i.p.) administration of GSK1059865 (1, 3, 10 mg/kg) dose-dependently reduces voluntary ethanol consumption: 10 mg/kg GSK1059865 decreases ethanol intake by 65% over 24 hours and lowers ethanol preference (ethanol/water volume ratio) from 0.75 to 0.22 [1]
2. GSK1059865 (3 mg/kg i.p.) in ethanol-dependent mice reduces ethanol relapse-like behavior (triggered by ethanol-associated cues) by 50%, with no effect on water or sucrose consumption (ruling out non-specific appetite suppression) [1]
3. In female Sprague-Dawley rats with binge eating behavior (induced by 14 days of restricted food access + palatable high-fat food), GSK1059865 (3, 10 mg/kg i.p.) dose-dependently reduces compulsive palatable food intake: 10 mg/kg decreases high-fat food consumption by 45% and binge episodes (≥1 g food consumed in 10 minutes) by 60% over 2 hours [2]
4. GSK1059865 (10 mg/kg i.p.) in female rats does not affect chow food intake or body weight over 7 days, indicating selective inhibition of reward-driven binge eating [2]
5. In a rat fMRI study using yohimbine (2 mg/kg i.p., a pharmacological stressor), GSK1059865 (3 mg/kg i.p.) inhibits yohimbine-induced activation of stress/reward brain regions: the blood oxygen level-dependent (BOLD) signal intensity in the central amygdala is reduced by 38%, and hypothalamic BOLD signal is decreased by 32% compared to vehicle controls [3]
6. GSK1059865 (3 mg/kg i.p.) in rats blunts yohimbine-induced increases in plasma corticosterone (a key HPA axis stress hormone) by 42% at 30 minutes post-yohimbine injection [3]

1. Human OX1R radioligand binding assay: Membranes were prepared from HEK293 cells stably expressing human OX1R. Membranes (50 μg protein/well) were incubated with [³H]orexin A (1 nM) and serial concentrations of GSK1059865 (0.01 nM–10 μM) in binding buffer (50 mM Tris-HCl, 5 mM MgCl₂, 0.1% BSA, pH 7.4) at 25°C for 90 minutes. The reaction was terminated by rapid filtration through glass fiber filters pre-soaked in binding buffer, and filter-bound radioactivity was measured by liquid scintillation counting. Non-specific binding was determined in the presence of 10 μM unlabeled orexin A, and Ki values were calculated using the Cheng-Prusoff equation [2]
2. OX1R functional Ca²⁺ mobilization assay: CHO cells expressing human OX1R were loaded with a calcium-sensitive fluorescent dye (4 μM) for 60 minutes at 37°C. GSK1059865 (0.1 nM–10 μM) was added 30 minutes before stimulation with orexin A (10 nM, EC80 for OX1R activation). Fluorescence intensity was measured every 2 seconds for 60 seconds using a fluorometer, and peak fluorescence responses were normalized to vehicle-treated controls to calculate IC50 values for inhibition [1][2]
3. CRF1 receptor binding assay: Membranes from CRF1-expressing HEK293 cells were incubated with [³H]CRF (1 nM) and GSK1059865 (0.1 nM–10 μM) in the same binding buffer for 90 minutes. Filtration and radioactivity measurement were performed as described above to assess potential binding to the CRF1 receptor [3]

1. OX1R-expressing CHO cell Ca²⁺ mobilization assay: CHO cells stably transfected with human OX1R cDNA were cultured in DMEM supplemented with 10% fetal bovine serum under 5% CO₂ at 37°C. Cells were seeded at 1×10⁴ cells/well in black-walled 96-well plates and allowed to adhere for 24 hours. After dye loading and GSK1059865 pretreatment, cells were stimulated with orexin A, and fluorescence was measured to quantify Ca²⁺ mobilization. Dose-response curves were fitted using nonlinear regression to determine the inhibitory potency of GSK1059865 [1][2]
2. Cell viability MTT assay: OX1R-expressing CHO cells and primary rat cortical neurons were seeded in 96-well plates (5×10³ cells/well) and treated with GSK1059865 (0.1 nM–10 μM) for 72 hours. MTT reagent (0.5 mg/mL) was added for 4 hours at 37°C, formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured using a microplate reader to calculate cell viability [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

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)

Solubility in Formulation 1: 3.33 mg/mL (7.63 mM) in 30 % SBE-β-CD (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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Solubility in Formulation 2: 5 mg/mL (11.46 mM) in 30% PEG300 70% (10% HP-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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.)