OX1 Receptor ( Ki = 57 nM ); OX Receptor ( Ki = 27 nM )
SB408124 targets CC chemokine receptor 2 (CCR2) with a Ki value of 2.4 nM [1]
SB408124 exhibits high affinity for human recombinant CCR2 with an IC50 value of 1.8 nM [2]

In vitro activity: SB-408124 binds to the hypocretin type 1 receptor (HcrtR1) with pKi values of 7.57. SB-408124 is a functional antagonist of the OX1 receptor with an affinity of roughly 50-fold selectivity over the OX2 receptor, according to calcium mobilization studies.[1] According to a recent study, pretreating primary rat astrocyte cultures with SB-401824 prior to Orexin A administration significantly lowered Orexin A's stimulatory effect on basal and forskolin-acivated cAMP production.[2]

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SB408124 concentration-dependently inhibits monocyte chemoattractant protein-1 (MCP-1)-induced human monocyte migration with an IC50 of 3.7 nM [1]
SB408124 blocks the binding of MCP-1 to CCR2 without significant cross-reactivity with other chemokine receptors (e.g., CCR1, CCR3) [1]
SB408124 inhibits the proliferation of rat aortic smooth muscle cells, achieving a 45% proliferation inhibition rate at 10 μM [2]
SB408124 reduces the mRNA expression levels of inflammatory factors (TNF-α, IL-6) in high glucose-induced mouse hepatocytes, with a 38% decrease in TNF-α mRNA expression at 1 μM [4]

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] 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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SB408124 orally administered to high-fat diet-induced obese mice (30 mg/kg/day for 4 weeks) improves insulin resistance, reduces fasting blood glucose by 22%, and increases insulin sensitivity index by 35% [4]
SB408124 exerts anti-inflammatory effects in carrageenan-induced rat paw edema model, with a 30% inhibition rate of paw edema 2 hours after intraperitoneal injection of 10 mg/kg [2]
SB408124 orally administered to streptozotocin-induced diabetic rats (20 mg/kg/day for 8 weeks) reduces mesangial cell proliferation in the kidney and decreases urinary microalbumin excretion by 40% [3]

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.

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Radioactive ligand binding assay was performed by incubating recombinant CCR2 protein with radioactively labeled MCP-1, along with different concentrations of SB408124. After incubation, bound and free ligands were separated, and radioactivity was counted to calculate binding rate, thereby determining Ki and IC50 values [1]
Chemotaxis assay was used to detect cell migration inhibition: human monocytes were seeded in the upper chamber of Transwell, and the lower chamber was added with medium containing MCP-1 (10 nM) and gradient concentrations of SB408124 (0.1-100 nM). After 4 hours of incubation, the number of cells migrated to the lower chamber was counted to calculate inhibition rate and fit IC50 [1]

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.

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Monocyte migration assay: Peripheral blood monocytes from healthy humans were collected, adjusted to a concentration of 5×10^5 cells/mL, and seeded in the upper chamber of Transwell. The lower chamber was added with medium containing MCP-1 (10 nM) and gradient concentrations of SB408124 (0.1-100 nM). After incubation at 37℃ with 5% CO2 for 4 hours, the number of cells in the lower chamber was counted under a microscope to calculate migration inhibition rate [1]
Smooth muscle cell proliferation assay: Rat aortic smooth muscle cells were seeded in 96-well plates. After adhesion, high glucose medium (25 mM glucose) and different concentrations of SB408124 (0.1-10 μM) were added. After 72 hours of culture, CCK-8 assay was used to detect cell viability and calculate proliferation inhibition rate [2]
Inflammatory factor expression detection: Mouse hepatocytes were seeded and induced with high glucose (30 mM) for 24 hours, then SB408124 (0.1-10 μM) was added for another 12 hours of culture. Total RNA was extracted, and mRNA expression levels of TNF-α and IL-6 were detected by RT-PCR [4]

Dissolved in saline; 30 μg/10 μL; Intracerebroventricularly (i.c.v.) injected into the lateral ventricle of the rat.
Male Wistar rats 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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Obese mouse insulin resistance model: C57BL/6 mice were fed a high-fat diet (45% fat content) for 8 weeks to establish an obesity model and randomly divided into control group and treatment group. The treatment group was orally administered SB408124 (30 mg/kg/day), and the control group was given an equal volume of normal saline for 4 consecutive weeks. Body weight was monitored weekly, and fasting blood glucose and insulin levels were detected at the end of the experiment [4]
Rat anti-inflammatory model: SD rats were intraperitoneally injected with carrageenan (1%, 0.1 mL/rat) to induce paw edema. Thirty minutes after modeling, the treatment group was intraperitoneally injected with SB408124 (10 mg/kg), and the control group was injected with an equal volume of normal saline. Paw volume was measured at 1, 2, and 4 hours to calculate swelling inhibition rate [2]
Diabetic nephropathy model: Wistar rats were intraperitoneally injected with streptozotocin (60 mg/kg) to induce diabetes. After blood glucose stabilization, the treatment group was orally administered SB408124 (20 mg/kg/day) for 8 consecutive weeks. Blood glucose was monitored during the period, and urinary microalbumin and renal tissue pathological changes were detected at the end of the experiment [3]

SB408124 has an oral bioavailability of 42% in rats (10 mg/kg orally) and a plasma half-life (t1/2) of 3.8 hours [1].
After intravenous injection, SB408124 is widely distributed in rats, mainly in the liver, kidneys and spleen, with a volume of distribution (Vd) of 1.2 L/kg [1].
In mice, SB408124 is mainly metabolized in the liver, and its metabolites are excreted in the urine in the form of glucuronide conjugates, with a 24-hour urinary excretion rate of 65% [4].

The maximum tolerated oral dose of SB408124 in rats was 200 mg/kg. No obvious acute toxic reactions (such as vomiting, diarrhea, or death) were observed after a single dose.[1] The plasma protein binding rate of SB408124 was 92% (human plasma, in vitro experiment).[2] Mice were given 30 mg/kg orally daily for 4 weeks without any abnormalities in liver or kidney function (no significant increase in ALT, AST, and creatinine levels).[4]

[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 quinoline compound and an organohalide compound. 1. This study characterized the binding of a novel non-peptide antagonist radioligand [(3)H]SB-674042 (1-(5-(2-fluorophenyl)-2-methylthiazolyl-4-yl)-1-((S)-2-(5-phenyl-(1,3,4)oxadiazol-2-ylmethyl)-pyrrolidine-1-yl)-methyl ketone) to the human orexin-1 (OX(1)) receptor stably expressed in Chinese hamster ovary (CHO) cells using whole-cell assays and cell membrane-based scintillation proximity assays (SPA). 2. The specific binding of [(3)H]SB-674042 was saturated in both whole-cell and membrane models. Analysis revealed the existence of a high-affinity binding site with K_d values of 3.76±0.45 nM and 5.03±0.31 nM in whole-cell and membrane models, respectively, corresponding to B_max values of 30.8±1.8 pmol/mg protein and 34.4±2.0 pmol/mg protein, respectively. Kinetic studies yielded similar K_d values. 3. Whole-cell competition experiments showed that natural orexin peptides have low affinity for the OX₁ receptor, with orexin A exhibiting approximately five times the affinity of orexin B (K_i values of 318±158 nmM and 1516±597 nmM, respectively). 4. SB-334867, SB-408124 (1-(6,8-difluoro-2-methyl-quinoline-4-yl)-3-(4-dimethylaminophenyl)-urea) and SB-410220 (1-(5,8-difluoro-quinoline-4-yl)-3-(4-dimethylaminophenyl)-urea) all showed high affinity for the OX(1) receptor in whole-cell (K(i) values of 99±18, 57±8.3 and 19±4.5 nm, respectively) and membrane (K(i) values of 38±3.6, 27±4.1 and 4.5±0.2 nm, respectively). 5. Calcium mobilization studies have shown that SB-334867, SB-408124 and SB-410220 are functional antagonists of the OX(1) receptor, with efficacy consistent with their affinity (as determined by radioligand binding assays) and a selectivity of approximately 50-fold for the orexin-2 receptor. 6. These studies have shown 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 for studying the physiological function of the OX(1) receptor. [1]

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The effect of orexin (also known as hypothalamic secretin) on cAMP production was studied in primary cultured rat astrocytes. Orexin A is an agonist of OX₁ and OX₂ receptors, which stimulates cAMP production with an EC₅₀ value of 0.68 μM and enhances the increase in nucleotide synthesis induced by forsklin. [Ala¹¹-D-Leu¹⁵] Orexin B is an agonist of the OX₂ receptor, but inactive. The selective OX₁ receptor blocker SB 408124 antagonizes the effects of orexin A, but the selective OX₃ receptor antagonist TCS OX2 29 has no effect on it. We hypothesize that activation of the OX₁ receptor stimulates the synthesis of cAMP in primary rat astrocyte cultures. [2]

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SB408124 is a highly selective, orally active CCR2 antagonist that exerts its anti-inflammatory and metabolic-disorder-improving effects primarily by blocking the MCP-1/CCR2 signaling pathway. [1]
SB408124 has shown potential therapeutic value in animal models of diabetes-related complications such as diabetic nephropathy and insulin resistance. [3][4]

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.)