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Biological Activity
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Description
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SB-408124 (Tocris-1963) is a non-peptide antagonist for OX1 with Ki of 57 nM and 27 nM in both whole cell and membrane, respectively. |
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Targets
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OX1 (whole cell) |
OX1 (membrane) |
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IC50 |
57 nM (Ki) |
27 nM (Ki) [1] |
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In Vitro
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SB-408124 binds hypocretin type 1 receptor (HcrtR1) with pKi values of 7.57. Calcium mobilization studies shows that SB-408124 is a functional antagonist of the OX1 receptor with a affinity of approximately 50-fold selectivity over the OX2 receptor. [1] A recent study indicates that pretreatment of primary cultures of rat astrocytes with SB-401824 before Orexin A administration significantly reduced the stimulatory action of Orexin A on both basal and forskolin-acivated cAMP production. [2] |
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In Vivo
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SB-408124 (30 μg/10 μL, administered intracerebroventricularly) decreases Orexin-A induced water intake in Wistar rats. Intracerebroventricularly administered Orexin-A (30 μg/10 μL) blocks the vasopressin (VP) level increase induced by either histamine or 2.5% NaCl administration, and this blocking effect is moderated by pretreatment with SB-408124. [3] Intracerebroventricular pretreatment with SB-408124 (50 mM, 5 μL/h) prevents Bicuculline (BIC)-induced increases in endogenous glucose production (EGP). [4] |
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Clinical Trials
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Features
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Protocol
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Kinase Assay
[1]
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| Whole cell binding assays |
After overnight culture in 96-well Packard Cultur plates, the medium is discarded and cells are incubated in buffer containing 150 mM NaCl, 20 mM HEPES and 0.5% bovine serum albumin (pH 7.4) for 60 minutes at 25 °C. Saturation studies are carried out by incubating cells with a range of concentrations of [3H]SB-408124 (0.2–24 nM); the total assay volume is 250 μL. Protein content is assayed by lysing cells with 0.1M NaOH and using the Bradford method with bovine serum albumin (BSA) as a standard. Association kinetic studies are performed by measuring the specific binding of [3H]SB-408124 (3 nM) at 1–60 minutes after addition of [3H]SB-408124. All assays are terminated by washing the cells three times with 250μL ice-cold phosphate-buffered saline. A volume of 100 μL of Microscint 40 is added to each well and the plate is left at room temperature for 2 hours. Cell-associated radioactivity is then measured using a Packard Topcount, with a count time of 2 minute/well. |
| Membrane-based SPA binding assays |
CHO-K1_OX1 cell membranes (75 μg/mL) are precoupled by shaking with wheatgerm-agglutinin polyvinyltoluene (WGA-PVT) scintillation proximity assay (SPA) beads (5 mg/mL) 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 hour. The bead-membrane suspension is centrifuged at 300× g and resuspended in the same volume of room temperature assay buffer. A volume of 100 μL of bead-membrane suspension is 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. Nonspecific binding is measured as that remaining in the presence of 3 μM SB-408124. Assay plates are shaken for 10 min and then incubated at room temperature for 4 hours before being counted on a Packard TopCount scintillation counter (count time 2 min/well). Saturation studies are carried out by incubating bead-membranes (equivalent to 7.5 μg protein/well and 2.5 mg beads/mL) with a range of concentrations of [3H]SB-674042 (0.1–20 nM). Protein content is assayed using the Bradford method using bovine serum albumin as a standard. Association kinetic studies are 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 and 2.5 mg beads/mL). For dissociation studies, bead-membranes are first incubated with [3H]SB-674042 (5 nM) for 30 min. Specific binding is then measured at 2–120 min after the addition of 3 μM SB-408124. Competition studies are performed by incubating bead-membranes (equivalent to 7.5 μg protein/well and 2.5 mg beads/mL) with [3H]SB-674042 (5 nM) and a range of concentrations of the test compound. |
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Animal Study
[3]
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| Animal Models |
Male Wistar rats |
| Formulation |
SB-408124 is dissolved in saline. |
| Doses |
30 μg/10 μL |
| Administration |
Intracerebroventricularly (i.c.v.) injected into the lateral ventricle of the rat. |
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References |
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[1] Langmead CJ, et al, Br J Pharmacol, 2004, 141(2) , 340-346.
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[2] Woldan-Tambor A, et al, Pharmacol Rep, 2011, 63(3), 717-723.
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[3] Kis Gk, et al, Pflugers Arch, 2012, 463(4), 531-536
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[4] Yi CX, et al, Diabetes, 2009, 58(9), 1998-2005.
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