Sch412348

Receptor binding is performed using membranes prepared from cells with recombinant expression of adenosine receptors as follows: human A_2A and HEK 293, rat A_2A and Chinese hamster ovary, human and rat A₁ and Chinese hamster ovary, and human A₃ and HEK 293. Radioligand competition binding assays are performed in 96-well plates in a total assay volume of 200 μL using a final test drug concentration range of between 0.1 and 3 μM. Membranes are diluted in assay buffer, pH 7.4 (A₁ and A_2A, Dulbecco's phosphate-buffered saline with 10 mM MgCl₂; A₃, 50 mM Tris-HCl, 120 mM NaCl, 10 mM MgCl₂). To remove endogenous adenosine from the membrane preparations, 4 U/mL adenosine deaminase is added to the membranes, which are then incubated at room temperature for 15 min. Radioligand is added to a final concentration of 0.5 ([³H]SCH 58261, A_2A), 1 ([³H]DPCPX, A₁), or 0.25 ([¹²⁵I]AB-MECA, A₃) nM. Nonspecific binding is defined by adding 100 nM CGS 15923 (A_2A), 100 nM NECA (A₁), or 100 nM DPCPX (A₃). Plates are incubated at room temperature with agitation for 1.5 h (A_2A and A₁) or 2 h (A₃). Membranes are filtered onto Packard GF-B filter plates and washed in ice-cold assay buffer using a Brandel cell harvester to separate bound and free radioligand. The plates are dried before addition of 45 μL of Microscint 20 to each well. IC₅₀ values are determining by fitting the displacement curves using an iterative curve-fitting program. K_i values are calculated using the Cheng-Prusoff equation^[1].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Rats^[1]
Male CD rats weighing 200 to 240 g are used for catalepsy and hypolocomotion studies. Sch412348 is administered orally in 50% polyethylene glycol 400 at a dose volume of 3 to 5 mL/kg. Sch412348 is administered in a volume of 1 mL/kg s.c.

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.