Bamirastine (TAK-427)

Kinase experiment:

The binding assay is performed using 96 well microplates, and 50 mM Tris-HCl containing 0.1% BSA, pH 7.4 is used as the assay buffer. Various concentrations of test compounds (50 μL/well), [3H] pyrilamine (22 nM, 25 μL/well, final 2.75 nM) and promethazine (80 μM, 25 μL/well, non-specific binding) or equal volumes of assay buffer are mixed, and the binding assay is initiated by the addition of the membrane suspension (5 μg protein/100 μL/well). The mixtures are incubated for 1 h at room temperature and the incubation is terminated by filtration over 0.3% polyethyleneimine treated UnifilterTM plates GF/C using a harvester. The UnifilterTM-plates are washed 3 times with 50 mM Tris-HCl buffer, pH 7.4, and dried completely. The radioactivity is counted by a TopCount system. Specific binding is defined as radioactivity bound after subtraction of nonspecific binding determined in the presence of promethazine. A Ki value (nM) is calculated, In order to characterize the inhibition of the H1 receptor binding by Bamirastine (TAK-427), saturation curves for specific binding of [3H] pyrilamine to the membranes expressing human histamine H1 receptors are investigated in the absence and presence of Bamirastine at 10 and 30 nM. The binding parameters are calculated from the Scatchard analysis of the saturation curves[1].

Animal experiment:

Mice[1] Male Crj: ICR mice (5 weeks old) are used. Bamirastine, Terfenadine and Epinastine in doses of 30, 100 and 300 mg/kg or vehicle (0.5% methylcellulose) are given orally. Behavior is observed for the first 2 h after drug administration.

References:

[1]. Fukuda S, et al. Characteristics of the antihistamine effect of TAK-427, a novel imidazopyridazine derivative. Inflamm Res. 2003 May;52(5):206-14.