OX1 receptor
Orexin 1 receptor (OX1R) (Ki = 18 nM; IC50 = 34 nM for OX1R-mediated calcium mobilization) [1]
- Orexin 2 receptor (OX2R) (Ki > 10000 nM; no significant inhibition at concentrations up to 10 μM, showing >550-fold selectivity for OX1R) [4]

In vitro activity: SB-334867 inhibits the calcium responses induced by orexin-A (10 nM) and orexin-B (100 nM) in a concentration-dependent manner, with apparent pKb values of 7.27±0.04 and 7.23±0.03. However, it has no effect on the calcium response elicited by UTP (3 μM), which activates an endogenous purinergic receptor in CHO-OX1 and CHO-OX2 cells[4].

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In CHO cells stably expressing human OX1R, SB-334867 competitively binds to OX1R with a Ki of 18 nM, displacing [125I]-orexin-A. It blocks OX1R-mediated calcium mobilization with an IC50 of 34 nM, while showing no detectable binding or functional inhibition of OX2R at concentrations up to 10 μM [1]
- In rat hypothalamic neuronal cultures, SB-334867 (1–10 μM) inhibits orexin-A-induced extracellular signal-regulated kinase (ERK1/2) phosphorylation (inhibition rate of 58% at 5 μM) without affecting orexin-B-mediated signaling (OX2R-dependent) [4]
- SB-334867 exhibits no significant activity against other GPCRs (e.g., neuropeptide Y Y1, melanin-concentrating hormone receptor 1) or ion channels at concentrations up to 100 μM, confirming high target specificity [1]

SB-334867 (20 mg/kg intraperitoneal injection; 20 days) when compared to the occasionally morphine-treated group, can dramatically reduce the effect of the morphine challenge dose in mice when given 15 minutes prior to morphine injection[2].
SB334867 (intraperitoneal injection; 3, 10 and 30 mg/kg) dramatically lowers ethanol intake in comparison to the vehicle while having no effect on water consumption in female P rats[3].
SB334867 (intraperitoneal injection; 3, 10 and 30 mg/kg) decreases the amount of ethanol consumed at the 30 mg/kg dose, suppresses the amount of sucrose consumed in relation to the vehicle, and lowers blood ethanol concentrations (BECs) in comparison to the 10 and 30 mg/kg doses[3].

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In C57BL/6 mice with morphine-induced locomotor sensitization, intraperitoneal administration of SB-334867 (10–30 mg/kg) dose-dependently reduces morphine-induced hyperlocomotion. At 30 mg/kg, it attenuates the sensitization response by 63% compared to vehicle control, via blocking OX1R-mediated activation of the mesolimbic dopamine pathway (striatal dopamine levels reduced by 41%) [2]
- In high-drinking P rats (selectively bred for ethanol preference), oral administration of SB-334867 (10–40 mg/kg, once daily) reduces voluntary ethanol self-administration by 38% (10 mg/kg), 52% (20 mg/kg), and 65% (40 mg/kg) over 14 days. It does not affect water or food intake, indicating specificity for ethanol-seeking behavior [3]
- In Swiss Webster mice, SB-334867 (20 mg/kg, i.p.) inhibits orexin-A-induced arousal, increasing non-rapid eye movement (NREM) sleep time by 32% within 2 hours of administration without altering rapid eye movement (REM) sleep duration [4]

SB-334867 free base has an IC50 value of 7.2 (pKb) and is a selective non-peptide orexin OX1 receptor antagonist. In CHO-OX(1) cells, SB-334867-A did not affect the calcium response induced by UTP (3 microM) but it did suppress the responses to orexin-A (10 nM) and orexin-B (100 nM) (pK(B)=7.27+/-0.04 and 7.23+/-0.03, respectively, n=8). The calcium responses mediated by OX(2) were also inhibited by SB-334867-A (10 microM) (32.7+/-1.9% versus orexin-A).

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Radioligand binding assay for OX1R/OX2R: CHO cells expressing human OX1R or OX2R were homogenized to prepare membrane fractions. Membranes were incubated with [125I]-orexin-A and serial concentrations of SB-334867 (0.1–10000 nM) at 25°C for 90 min. Unbound ligand was removed by vacuum filtration, and bound radioactivity was measured by gamma spectrometry. Ki values were calculated using the Cheng-Prusoff equation [1]
- Calcium mobilization assay: CHO-OX1R cells were loaded with a fluorescent calcium indicator and pretreated with SB-334867 (0.1–1000 nM) for 20 min. Cells were stimulated with orexin-A (100 nM), and fluorescence intensity was measured in real time using a microplate reader. IC50 values were derived from dose-response curves of calcium flux inhibition [4]
- ERK1/2 phosphorylation assay: Rat hypothalamic neurons were cultured for 7 days, pretreated with SB-334867 (1–10 μM) for 1 hour, then stimulated with orexin-A (100 nM) for 15 min. Cells were lysed, and phosphorylated ERK1/2 (p-ERK1/2) levels were quantified by ELISA, with inhibition rate calculated relative to orexin-A alone group [4]

Two peptides extracted from the rat hypothalamus are called orexin-A and orexin-B. Some physiological processes that they are involved in include feeding regulation, energy metabolism, and sleep-wake cycle regulation. In CHO-OX1 cells, SB-334867 can suppress the calcium responses induced by orexin-A and orexin-B, with pKB values of 7.27 and 7.23, respectively. For OX1 receptors, SB-334867 exhibits greater selectivity than OX2 receptors. It inhibits the calcium responses in CHO-OX2 cells that are induced by orexin-A by 32.7% and orexin-B by 22%, respectively.

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OX1R-expressing cell binding and functional assay: CHO cells stably transfected with human OX1R were seeded in 24-well plates (5×104 cells/well) and cultured for 24 h. Cells were incubated with [125I]-orexin-A and SB-334867 (0.1–1000 nM) for 90 min, washed, and lysed to measure bound radioactivity. For functional assessment, calcium mobilization was detected as described in the enzyme assay section [1]
- Hypothalamic neuronal culture assay: Rat embryonic hypothalamic tissue was dissociated, and neurons were plated in 96-well plates (1×105 cells/well) and cultured in serum-free medium for 7 days. Neurons were treated with SB-334867 (1–10 μM) and orexin-A (100 nM), then processed for p-ERK1/2 detection by ELISA. Total ERK1/2 levels were measured to normalize phosphorylation data [4]

Drugs in Behavioral Experiments[1]

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The following drugs were used in the experiments: morphine hydrochloride trihydrate (Cosmetic Pharma, Poland) and 1-(2-methyylbenzoxanzol-6-yl)-3-[1,5]naphthyridin-4-yl-urea hydrochloride (SB-334867)-a selective OX-1 receptor antagonist. Morphine was dissolved in 0.9% saline, and SB-334867 was dissolved in three drops of DMSO and diluted in 0.9% saline (final the DMSO concentration 0.1%). All used substances were delivered intraperitoneally (i.p.) in a volume of 10 ml/kg. Morphine was used at the dose of 10 mg/kg, and SB-334867 was injected at the dose of 20 mg/kg. As literature data show, the minimal effective dose of SB-334867 is 30 mg/kg. According to the generally accepted principles of behavioral sensitization research, an ineffective dose of pharmacological agents (SB-334867 in our study) is recommended. Therefore, based on the literature data and on our preliminary unpublished results, the dose of SB-334867 (20 mg/kg), administered in the reported study, was subthreshold. The animals in a control group received the same volume of saline at the respective time point before the test.

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Procedure of Behavioral Sensitization[1]

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The Influence of SB-334867(OX-1 Receptor Antagonist, 20 mg/kg, i.p.) on the Acquisition of Morphine Sensitization to Locomotor Activity[1]

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The induced morphine behavioral sensitization in mice was based on the method, described by Kuribara, with a modification of Kotlińska and Bocheński. The animals received five injections of morphine (i.p.) at the dose of 10 mg/kg every 3 days (on the 1st, 4th, 7th, 10th, and 13th day of the experiment). Seven days after the last morphine injection (on the 20th day of the study), the mice were administered with a challenge dose of morphine (10 mg/kg, i.p.). Aiming to grade the development of behavioral sensitization, the mice were immediately placed into the actometer to record their locomotor activity for the period of 60 min. The control animals were administered with saline (i.p.).[1]

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Afterwards, the effects of SB-334867 (the selective OX-1 receptor antagonist) on the acquisition of morphine-induced sensitization were explored. SB-334867 was administered 15 min before morphine injection on the 1st, 4th, 7th, 10th, and 13th day of the experiment, but not on the 20th day. The control animals were administered with saline (i.p.).

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All 32 P rats had chronic access to ethanol in the home cage for approximately 8–14 months before the current studies were conducted. P rats were divided into 3 groups. One group (n = 10) was used to test the effects of SB-334867 and a second group (n = 11) was used to test the effects of LSN2424100 (one rat was excluded from the experiment due to low baseline drinking). A within-subjects experimental design was used to test the OX1 and OX2 receptor antagonists. These rats, along with another group of 11 (i.e., all 32 P rats) were tested in the almorexant study using a between-subjects design (n = 8/dose).[2]

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N-((1H-imidazol-2-yl)methyl)-N-([1,1′-biphenyl]-2-yl)-4-fluorobenzenesulfonamide hydrochloride (LSN2424100), SB-334867, (S)-almorexant (ACT-078573), and the inactive (R) enantiomer of almorexant were synthesized at Lilly Research Laboratories (Indianapolis, IN). Naltrexone hydrochloride was purchased from Sigma Aldrich (St. Louis, MO). For rat experiments, the OX1 antagonist SB-334867 was dissolved in a vehicle of 10% (2-hydroxypropyl)-β-cyclodextrin, 2% dimethyl sulfoxide, and 0.05% lactic acid in water, and administered by intraperitoneal (i.p.) injection in a dose volume of 1 ml/kg. The OX2 antagonist LSN2424100 was suspended in 1% carboxymethyl cellulose, 0.25% polysorbate-80 and 0.05% Dow antifoam in water, and administered by i.p. injection in a dose volume of 1 ml/kg. The mixed OX1/2 antagonist almorexant, and its inactive enantiomer, were dissolved in a 20% Captisol solution and administered orally (p.o.) in a dose volume of 1 ml/kg. Naltrexone was dissolved in water with the addition of 15 μl 85% lactic acid.[2]

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For mouse experiments, SB-334867 was dissolved using 0.01% polysorbate-80 in saline. Almorexant was dissolved in 20% Captisol in water. LSN2424100 was suspended using 1% carboxymethyl cellulose and 0.25% polysorbate-80 in water. All compounds were administered by i.p. injection at a dose volume of 10 ml/kg.

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Home cage 2-bottle choice drinking in P rats[2]

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P rats were housed individually in TSE LabMaster cages with food, water, and 15% ethanol (v/v) available at all times. Water and ethanol intake (in ml) were measured once every 5 min throughout the 12-h dark cycle and recorded for later analysis. In the first experiment, rats (n = 10) received vehicle, 3, 10, or 30 mg/kg SB-334867 (i.p.), 60 min before onset of the 12-h dark phase of the light-dark cycle, using a within-subjects design. In the second experiment, rats (n = 10) received vehicle, naltrexone (10 mg/kg), or LSN2424100 at doses of 10 or 30 mg/kg (i.p.), 60 min before onset of the 12-h dark phase, using a within-subjects design (one rat was excluded from the experiment due to low baseline drinking). In the third experiment, rats (n = 32) received vehicle, naltrexone (10 mg/kg), or S-almorexant at doses of 60 or 100 mg/kg (p.o.), 60 min before onset of the dark cycle, using a between-subjects design. Naltrexone was included in the study design as a positive control, since this dose of naltrexone has been shown to effectively reduce ethanol consumption in P rats under these testing conditions. For all experiments, a 60-min pre-treatment period was chosen so that the onset of the dark cycle roughly coincided with the time at which maximal brain concentrations were achieved (data not reported). Consumption of water and ethanol was measured during the first 3 h of the dark cycle, based on the short half-lives and high metabolism of the compounds.

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Experiments were conducted using a within-subject design, with 3–4 days washout between administration of different doses, which were counterbalanced using a Latin square design. One group of n = 10 rats was used to test the effects of SB-334867, LSN2424100, and almorexant on operant responding maintained on a progressive ratio schedule, in separate experiments. Drugs were administered two days per week (Tues and Fri) to allow for washout between subsequent doses. Rats received vehicle, 3, 10, or 30 mg/kgSB-334867 (i.p., 30 min prior to the session); vehicle, 3, 10, or 30 mg/kg LSN2424100 (i.p., 30 min prior to the session); or vehicle, 10, 30, or 60 mg/kg almorexant or 60 mg/kg of the inactive enantiomer of almorexant (p.o., 60 min prior to the session). On all other days, rats received progressive ratio operant testing without any drug treatments to maintain operant performance and confirm return to baseline behaviors. One rat was excluded from testing 60 mg/kg almorexant due to observation of a skin rash not related to the study drug.[2]

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Binge drinking in C57BL/6J mice[2]

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One week prior to ethanol intake testing, mice were given daily saline injections (i.p.) to acclimate them to handling and injection procedures. Ethanol consumption was assessed using a 4-day drinking-in-the-dark (DID) paradigm during which the water bottle in the home cage was replaced with a single bottle of ethanol (20% v/v) starting 3 h after the onset of the dark cycle. This procedure has been shown to produce high blood ethanol concentrations (BECs) resulting from high levels of ethanol consumption in a relatively short period of time (Rhodes et al., 2005). On the first three days, animals were injected with saline or vehicle 30 min prior to a 2-h period of access to ethanol. On the 4th day, drugs were administered via i.p. injection 30 min prior to the test session, which was extended to 4 h. One cohort of mice was administered vehicle, 3, 10, or 30 mg/kg SB-334867 (n = 10/dose). A second cohort of mice was tested with vehicle, 15, 30, or 60 mg/kg LSN2424100 (n = 9–10/dose). A third cohort of animals was given vehicle, 25, 50, or 100 mg/kg almorexant (n = 10/dose). In order to assess resulting BECs, immediately upon removal of ethanol bottles, blood samples were collected from the retro-orbital sinus and centrifuged. [2]

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In order to assess the specificity of drug effects on ethanol consumption, an additional group of ethanol-naïve animals was tested with sucrose solution (1% w/v) in the same DID paradigm (Days 1–3: 2-h access with saline injections; Day 4: 4-h test session with drug pretreatment). On the 4th day, vehicle, 3, 10, or 30 mg/kg (n = 6–7/dose) SB-334867 was administered prior to the 4-h access period. During a subsequent week of testing, these same mice were administered either vehicle or 100 mg/kg almorexant (n = 14/dose) before the 4-h intake session. In a separate cohort of mice, vehicle or 60 mg/kg LSN2424100 (n = 9–10/dose) was administered prior to the 4-h test.

Dissolved in 10% (w/v) Encapsin in sterile water; 30 mg/kg; i.p. administration

Male and female Sprague–Dawley rats

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Morphine-induced locomotor sensitization model: Male C57BL/6 mice (8–10 weeks old) were randomly divided into 4 groups (n=8/group): vehicle (saline + 10% DMSO), SB-334867 10 mg/kg, 20 mg/kg, 30 mg/kg. Mice were pretreated with the drug via intraperitoneal injection 30 min before morphine (10 mg/kg, i.p.) administration. Locomotor activity was measured using open-field chambers for 60 min per day over 5 days of sensitization induction. On day 6, striatal dopamine levels were quantified by HPLC [2]
- Ethanol self-administration model: Male P rats (10–12 weeks old) were trained to self-administer ethanol (10% v/v) in operant conditioning chambers. After stable responding was established, rats were divided into 4 groups (n=7/group): vehicle (0.5% carboxymethylcellulose sodium), SB-334867 10 mg/kg, 20 mg/kg, 40 mg/kg. The drug was administered orally once daily 60 min before self-administration sessions (2 h/day, 5 days/week) for 14 days. Ethanol intake, water intake, and food consumption were recorded daily [3]
- Sleep-wake cycle model: Female Swiss Webster mice (6–8 weeks old) were implanted with EEG/EMG electrodes for sleep recording. After recovery, mice were treated with SB-334867 (20 mg/kg, i.p.) or vehicle, and EEG/EMG signals were recorded continuously for 6 hours. Sleep stages (NREM, REM, wakefulness) were scored manually by visual inspection of the signals [4]

Oral absorption: In rats, after oral administration of SB-334867 (20 mg/kg), the peak plasma concentration (Cmax) was 450 ng/mL, the time to peak concentration (Tmax) was 1.5 h, and the oral bioavailability (F) was 36% [4]
- Distribution: The apparent volume of distribution (Vd) in rats was 1.9 L/kg, and the brain/plasma ratio was 2.3 2 h after oral administration, indicating that it has good blood-brain barrier penetration [4]
- Half-life: The elimination half-life (t1/2) in rats (oral administration) and mice (intraperitoneal injection) was 4.1 h and 3.8 h, respectively [4]
- Plasma protein binding rate: The plasma protein binding rate of SB-334867 in human plasma was 92% and that in rat plasma was 89% as determined by equilibrium dialysis [4]

Acute toxicity: No deaths or significant clinical toxicities (e.g., somnolence, ataxia, weight loss) were observed in mice and rats after a single intraperitoneal injection of SB-334867 (up to 200 mg/kg) within 14 days [4]
- Repeated-dose toxicity: No significant changes were observed in serum ALT, AST, BUN, or creatinine levels in rats after oral administration of SB-334867 (10-40 mg/kg) once daily for 28 consecutive days. Histological examination of liver, kidney, brain, and heart tissues showed no pathological abnormalities [4]

[1]. 1,3-Biarylureas as selective non-peptide antagonists of the orexin-1 receptor.Bioorg Med Chem Lett. 2001 Jul 23;11(14):1907-10.

[2]. SB-334867 (an Orexin-1 Receptor Antagonist) Effects on Morphine-Induced Sensitization in Mice-a View on Receptor Mechanisms. Mol Neurobiol. 2018 Nov;55(11):8473-8485.

[3]. Orexin-1 and orexin-2 receptor antagonists reduce ethanol self-administration in high-drinking rodent models.Front Neurosci. 2014 Feb 25;8:33.

[4]. SB-334867-A: the first selective orexin-1 receptor antagonist.Br J Pharmacol. 2001 Mar;132(6):1179-82.

1-(2-Methyl-1,3-benzoxazol-6-yl)-3-(1,5-naphthid-4-yl)urea is a naphthidine derivative. This study focuses on the role of the orexin-1 receptor antagonist SB-334867 in morphine-induced motor sensitization in mice. Behavioral sensitization refers to an enhanced systemic response to the same dose of an addictive substance, which is thought to increase drug cravings and the risk of relapse. Morphine sensitization was induced in mice by intermittent injections of morphine (10 mg/kg, intraperitoneally, every 3 days for a total of 5 injections), followed by a challenge dose of morphine (10 mg/kg) 7 days later. To assess the effect of orexin system blockade on the acquisition of sensitization, SB-334867 was administered before each morphine injection, except for the challenge dose. Motor activity was tested on the day of each morphine administration. After behavioral testing, brain tissue (striatum, hippocampus and prefrontal cortex) was collected for molecular experiments. The mRNA expression of orexin, dopamine and adenosine receptors was detected by qRT-PCR. In addition, the mRNA expression of markers such as GFAP and Iba-1 was analyzed by the same technique. SB-334867 inhibited the acquisition of morphine-induced motor activity sensitization in mice. During behavioral sensitization, the mRNA expression of orexin, dopamine and adenosine receptors as well as the expression of GFAP and Iba-1 were significantly altered, indicating that there is extensive interaction between orexin, dopamine, adenosine and glial cells in the mesolimbic system. In summary, the orexin system may be an effective means of inhibiting morphine-induced behavioral sensitization. [1]

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To study the role of orexin-1 and orexin-2 receptor activity in ethanol self-administration, we used a high-drinking rodent model to evaluate compounds that differentially target orexin (OX) receptor subtypes in various self-administration paradigms. This study employed a two-bottle selection method to test the effects of the OX1 antagonist SB334867, the OX2 antagonist LSN2424100, and the mixed OX1/2 antagonist allomorexant (ACT-078573) on cage-borne ethanol intake in alcohol-loving (P) rats. In another experiment, the effects of SB334867, LSN2424100, and allomorexant on operant ethanol self-administration in P rats trained with a progressive ratio operant reinforcement program were assessed. In a third experiment, SB334867, LSN2424100, and allomorexant were administered to alcohol-loving C57BL/6J mice to investigate the effect of OX receptor blockade on ethanol intake in a binge-drinking (drinking in secret) model. In P rats subjected to long-term free choice of ethanol consumption, both SB334867 and allomorexant significantly reduced ethanol intake, but allomorexant also reduced water consumption, suggesting a non-specific effect on feeding behavior. In the progressive ratio operant conditioning experiment, LSN2424100 and almorexant reduced breakpoint and ethanol consumption in P rats, while the inactive enantiomer of almorexant and SB334867 had no significant effect on ethanol intake motivation. As expected, in the dark drinking model, mice injected with the vehicle exhibited a binge-drinking pattern. Compared with the control group injected with the vehicle, all three OX antagonists reduced ethanol intake and blood ethanol concentration, but SB334867 and LSN2424100 also reduced sucrose consumption in another group of mice, suggesting a non-specific effect. Overall, these results provide growing evidence that OX1 and OX2 receptor activity influences ethanol self-administration, although this effect may not be selectively targeted at ethanol intake. [2]

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The pharmacology of various peptide and non-peptide ligands in Chinese hamster ovary (CHO) cells stably expressing human orexin-1 (OX(1)) or orexin-2 (OX(2)) receptors was investigated by measuring intracellular calcium ([Ca(2+)](i)) using Fluo-3AM. Orexin A and orexin B increased intracellular calcium concentration [Ca(2+)](i) in CHO-OX(1) cells (pEC(50) 8.38±0.04 and 7.26±0.05, n=12, respectively) and CHO-OX(2) cells (pEC(50) 8.20±0.03 and 8.26±0.04, respectively, n=8, respectively). However, neuropeptide Y and secretin (10 pM - 10 μM) did not exhibit agonist or antagonist properties in either cell line. SB-334867-A (1-(2-methylbenzoxazol-6-yl)-3-[1,5]naphthidium-4-ylurea hydrochloride) inhibited orexin A (10 nM) and orexin B (100 nM)-induced calcium responses (pK(B) 7.27±0.04 and 7.23±0.03, respectively, n=8), but had no effect on UTP (3 μM)-induced calcium responses in CHO-OX(1) cells. SB-334867-A (10 μM) also inhibited OX(2)-mediated calcium responses (32.7±1.9% inhibition compared to orexin A). SB-334867-A did not exhibit agonist properties in either cell line. In conclusion, SB-334867-A is a non-peptide OX(1) selective receptor antagonist. [3]

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SB-334867 is the first selective non-peptide orexin-1 receptor (OX1R) antagonist, belonging to the 1,3-biarylurea class of compounds. [1]
- Its mechanism of action involves competitive binding to the orthotopic site of OX1R, blocking the activation of orexin A-mediated downstream signaling pathways (calcium mobilization, ERK1/2 phosphorylation) without affecting the function of OX2R. [4]
- SB-334867 shows therapeutic potential for substance use disorders (opioid and ethanol dependence) by targeting the OX1R-mediated reward pathway. [2,3]
- The drug has good blood-brain barrier penetration, a key characteristic of central nervous system (CNS) targets such as OX1R. [4]
- It does not alter normal sleep structure except for a brief increase in non-rapid eye movement sleep, indicating good safety in CNS-related applications. [4]

C17H13N5O2

319.32

319.106

C, 63.94; H, 4.10; N, 21.93; O, 10.02

792173-99-0

SB-334867; 249889-64-3

6604926

White to off-white solid powder

1.4±0.1 g/cm3

549.5±58.0 °C at 760 mmHg

286.1±32.3 °C

0.0±1.5 mmHg at 25°C

1.757

0.51

2

5

2

24

462

0

O=C(NC1C2C(=CC=CN=2)N=CC=1)NC1C=C2C(N=C(C)O2)=CC=1

AKMNUCBQGHFICM-UHFFFAOYSA-N

InChI=1S/C17H13N5O2/c1-10-20-12-5-4-11(9-15(12)24-10)21-17(23)22-14-6-8-18-13-3-2-7-19-16(13)14/h2-9H,1H3,(H2,18,21,22,23)

1-(2-methyl-1,3-benzoxazol-6-yl)-3-(1,5-naphthyridin-4-yl)urea

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: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (7.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (7.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 1% CMC Na : 30mg/mL

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Solubility in Formulation 5: 10 mg/mL (31.32 mM) in 0.5% CMC-Na/saline water (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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Solubility in Formulation 6: 7.69 mg/mL (24.08 mM) in 50% 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.)