OX1 Receptor ( Ki = 57 nM ); OX Receptor ( Ki = 27 nM )

Corticoliberin (CRF) isn't only regulates hypothalamic-pituitary-adrenal axis activity, but also functions as a neurotransmitter in extrahypothalamic brain regions like amygdala, implicated in the emotional responses to stress. The CRF system provides an input to orexin neurons and can modulate the activity of orexinergic neurons in stress response. Some data showed the role of orexin-A in extinction of aversive memory. The orexin system was shown to participate in stress-induced behavior connected with the extended amygdala structures, like central nucleus of the amygdala. The objective was to study the effects of orexin-A antagonist SB-408124 in rats after predator-induced stress using behavioral tests and its effects on CRF level in amygdala. In this study 30 male Wistar rats were used. The animals received an intranasally selective antagonist of Orexin receptor 1 type SB-408124. Posttraumatic stress disorder was modelled by single predator exposure. A group of 10-12 rats were placed in a terrarium with an indian python. 7 days after exposure to the predator, the behavior of animals was tested in the Open Field and Elevated Cross-Maze tests. Free motor activity of animals was studied in the "open field" test. To assess stress, we used the "elevated cross-maze " test. CRF concentrations in brain structures were measured by solid-phase ELISA using the Corticotropin Releasing Factor (CRF) test system. In the group of stressed rats receiving intranasally SB-408124, the time of stay in the light arm was restored, but did not reach the control values, the number of runs was restored to the control level, and the number of grooming acts increased in comparison with both the control group and the stressed animals. In the "open field" in the group of stressed rats receiving saline solution, the number of sniffs and rearing were decreased, but the number of peeks into holes was increased. In the group of stressed rats receiving SB-408124 20 µg intranasally, the number of sniffs was increased and the number of hole peeking decreased in comparison with the stressed rats receiving saline solution. The CRF level in the homogenates of amygdala in stressed rats was lower (0.44±0.07 ng/mg protein vs. 0.61±0.01 ng/mg in the control group). In the intranasal administration of SB-408124 group this decrease was not recorded and the CRF level in the amygdala was 0.57±0.01 pg/mg protein. Orexin A antagonist SB-408124 reduced anxiety after psychotraumatic exposure. Predator induced acute psychotraumatic exposure decrease CRF level in the rat's amygdala. Intranasal administration of selective orexin 1 receptor antagonist SB-408124 restored it closely to normal and has an anxiolytic effect on animal behaviour.[1]

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Furthermore, bilateral PVN microinjection of the OX1R antagonist SB-408124 resulted in a greater reduction in MAP in HS intake (-16 ± 5 mmHg) compared with NS-fed (-4 ± 4 mmHg) anesthetized Dahl S rats. These results suggest that elevated PVN OX1R activation may contribute to SSHTN by enhancing AVP signaling.[2]

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SB-408124 (30 μg/10 μL) reduces the amount of water induced by Orexin-A in Wistar rats. Orexin-A (30 μg/10 μL) given intracerebroventricularly inhibits the rise in vasopressin (VP) levels brought on by either histamine or 2.5% NaCl administration; this inhibitory effect is mitigated by SB-408124 pretreatment. [3] SB-408124 (50 mM, 5 μL/h) administered intracerebroventricularly precludes increases in endogenous glucose production (EGP) brought on by bicuculline (BIC). [4]

SB-408124 is a non-peptide antagonist that shows 50-fold selectivity over OX2 receptor and has a Ki of 57 nM and 27 nM in whole cell and membrane, respectively, for the OX1 receptor.

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[3H]SB-674042 whole cell binding assays[1]
After overnight culture in 96-well Packard Cultur plates, the medium was discarded and cells were incubated in buffer containing 150 mM NaCl, 20 mM HEPES and 0.5% bovine serum albumin (pH 7.4) for 60 min at 25°C. Saturation studies were carried out by incubating cells with a range of concentrations of [3H]SB-674042 (0.2–24 nM); the total assay volume was 250 μl. Protein content was assayed by lysing cells with 0.1 M NaOH and using the Bradford method (Bradford, 1976) with bovine serum albumin (BSA) as a standard.
Association kinetic studies were performed by measuring the specific binding of [3H]SB-674042 (3 nM) at 1–60 min after addition of [3H]SB-674042. For dissociation studies, cells were first incubated with [3H]SB-674042 (3 nM) for 60 min. Specific binding was then measured at 2–120 min after the addition of 3 μM SB-408124. Competition studies were performed by incubating cells with [3H]SB-674042 (3 nM) and a range of concentrations of the test compound. All assays were terminated by washing the cells three times with 250 μl ice-cold phosphate-buffered saline. A volume of 100 μl of Microscint 40 was added to each well and the plate was left at room temperature for 2 h. Cell-associated radioactivity was then measured using a Packard Topcount, with a count time of 2 min well−1.

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[3H]SB-674042 membrane-based SPA binding assays[1]
CHO-K1_OX1 cell membranes (75 μg ml−1) were precoupled by shaking with wheatgerm-agglutinin polyvinyltoluene (WGA-PVT) scintillation proximity assay (SPA) beads (5 mg ml−1) in buffer containing 25 mM HEPES, 2.5 mM MgCl2, 0.5 mM EDTA and 0.025% bacitracin (pH 7.4) at 4°C for 1 h. The bead-membrane suspension was centrifuged at 300 × g and resuspended in the same volume of room temperature assay buffer. A volume of 100 μl of bead-membrane suspension was incubated with [3H]SB-674042 (5 nM) in a total assay volume of 200 μl in a 96-well Packard Optiplate to give a final protein concentration of 7.5 μg well−1. Nonspecific binding was measured as that remaining in the presence of 3 μM SB-408124. Assay plates were shaken for 10 min and then incubated at room temperature for 4 h before being counted on a Packard TopCount scintillation counter (count time 2 min well−1).
Saturation studies were carried out by incubating bead-membranes (equivalent to 7.5 μg protein well−1 and 2.5 mg beads ml−1) with a range of concentrations of [3H]SB-674042 (0.1–20 nM). Protein content was assayed using the Bradford method (Bradford, 1976) using bovine serum albumin as a standard. Association kinetic studies were performed by measuring specific binding of [3H]SB-674042 (5 nM) at 1–30 min after addition of bead-membranes (equivalent to 7.5 μg protein well−1 and 2.5 mg beads ml−1). For dissociation studies, bead-membranes were first incubated with [3H]SB-674042 (5 nM) for 30 min. Specific binding was then measured at 2–120 min after the addition of 3 μM SB-408124. Competition studies were performed by incubating bead-membranes (equivalent to 7.5 μg protein well−1 and 2.5 mg beads ml−1) with [3H]SB-674042 (5 nM) and a range of concentrations of the test compound.

SB-408124 has a pKi of 7.57 when it comes to binding the hypocretin type 1 receptor (HcrtR1). According to studies on calcium mobilization, SB-408124 functions as a functional antagonist of the OX1 receptor and has an affinity that is roughly 50 times more selective than the OX2 receptor. According to a recent study, the stimulatory action of Orexin A on basal and forskolin-acivated cAMP production was significantly reduced when primary cultures of rat astrocytes were pretreated with SB-401824 prior to Orexin A administration.

PVN injections were performed as previously described. Animals were placed in a stereotaxic head frame, and the skull was leveled between the bregma and lambda for PVN injections. A small piece of the skull was removed so that a single-barreled glass microinjector pipette could be lowered vertically into the PVN. The stereotaxic coordinates for PVN injections were as follows: 1.2–1.6 mm caudal to the bregma, 0.5–0.7 mm lateral to the midline, and 7.0–7.4 mm ventral to the dura. After a 20-min baseline period, SB-408124 (30 pmol) was microinjected into the PVN bilaterally in a volume of 50 nl/side with a pneumatic pump (World Precision Instruments). The OX1R antagonist SB-408124 was dissolved in DMSO. The interval between two bilateral injections was ∼5 min.[Georgian Med News. 2019 May;(290):127-131.]

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Dissolved in saline; 30 μg/10 μL; Intracerebroventricularly (i.c.v.) injected into the lateral ventricle of the rat.

Male Wistar rats

[1]. Br J Pharmacol . 2004 Jan;141(2):340-6.

[2]. Pharmacol Rep . 2011;63(3):717-23.

[3]. Pflugers Arch . 2012 Apr;463(4):531-6.

[4]. Diabetes . 2009 Sep;58(9):1998-2005.

1-(6,8-difluoro-2-methyl-4-quinolinyl)-3-[4-(dimethylamino)phenyl]urea is a member of quinolines and an organohalogen compound.
1. This study characterises the binding of a novel nonpeptide antagonist radioligand, [(3)H]SB-674042 (1-(5-(2-fluoro-phenyl)-2-methyl-thiazol-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidin-1-yl)-methanone), to the human orexin-1 (OX(1)) receptor stably expressed in Chinese hamster ovary (CHO) cells in both a whole cell assay and in a cell membrane-based scintillation proximity assay (SPA) format. 2. Specific binding of [(3)H]SB-674042 was saturable in both whole cell and membrane formats. Analyses suggested a single high-affinity site, with K(d) values of 3.76+/-0.45 and 5.03+/-0.31 nm, and corresponding B(max) values of 30.8+/-1.8 and 34.4+/-2.0 pmol mg protein(-1), in whole cell and membrane formats, respectively. Kinetic studies yielded similar K(d) values. 3. Competition studies in whole cells revealed that the native orexin peptides display a low affinity for the OX(1) receptor, with orexin-A displaying a approximately five-fold higher affinity than orexin-B (K(i) values of 318+/-158 and 1516+/-597 nm, respectively). 4. SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinolin-4-yl)-3-(4-dimethylamino-phenyl)-urea) all displayed high affinity for the OX(1) receptor in both whole cell (K(i) values 99+/-18, 57+/-8.3 and 19+/-4.5 nm, respectively) and membrane (K(i) values 38+/-3.6, 27+/-4.1 and 4.5+/-0.2 nm, respectively) formats. 5. Calcium mobilisation studies showed that SB-334867, SB-408124 and SB-410220 are all functional antagonists of the OX(1) receptor, with potencies in line with their affinities, as measured in the radioligand binding assays, and with approximately 50-fold selectivity over the orexin-2 receptor. 6. These studies indicate that [(3)H]SB-674042 is a specific, high-affinity radioligand for the OX(1) receptor. The availability of this radioligand will be a valuable tool with which to investigate the physiological functions of OX(1) receptors.[1]

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The effects of orexins, which are also named hypocretins, on cAMP formation were examined in primary cultures of rat astrocytes. Orexin A, an agonist of OX₁ and OX₂ receptors, stimulated cAMP production with an EC₅₀ value of 0.68 μM and potentiated the forskolin-induced increase in the nucleotide synthesis. [Ala¹¹-D-Leu¹⁵]orexin B, an agonist of OX₂ receptors, was inactive. The effects of orexin A were antagonized by SB 408124, a selective blocker of OX₁ receptors, but were not affected by TCS OX2 29, a selective antagonist of OX₃ receptors. We hypothesized that the activation of OX₁ receptors stimulated cAMP synthesis in primary rat astrocyte cultures.[2]

C19H18F2N4O

356.37

356.144

C, 64.04; H, 5.09; F, 10.66; N, 15.72; O, 4.49

288150-92-5

SB-408124 Hydrochloride; 1431697-90-3

4331799

White to off-white solid powder

1.4±0.1 g/cm3

430.3±45.0 °C at 760 mmHg

214.0±28.7 °C

0.0±1.0 mmHg at 25°C

1.697

4.53

2

5

3

26

484

0

FC1=C([H])C(=C([H])C2C1=NC(C([H])([H])[H])=C([H])C=2N([H])C(N([H])C1C([H])=C([H])C(=C([H])C=1[H])N(C([H])([H])[H])C([H])([H])[H])=O)F

JTARFZSNUAGHRB-UHFFFAOYSA-N

InChI=1S/C19H18F2N4O/c1-11-8-17(15-9-12(20)10-16(21)18(15)22-11)24-19(26)23-13-4-6-14(7-5-13)25(2)3/h4-10H,1-3H3,(H2,22,23,24,26)

1-(6,8-difluoro-2-methylquinolin-4-yl)-3-[4-(dimethylamino)phenyl]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 (7.02 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 2: 30% propylene glycol, 5% Tween 80, 65% D5W: 30mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)