Four ICI-162846 doses (0.5, 1.0, 2.5 and 5.0 mg) were given as a single tablet in randomized double-blind fashion

Chinese hamster ovary (CHO) cells stably expressing a cyclic AMP response element-secreted placental alkaline phosphatase (CRE-SPAP) reporter gene were secondarily transfected with the human histamine H2 receptor. Cells were grown to confluence in 24-well plates. The media were removed and the cells pre-labelled for 3 h with 3H-adenine by incubation with 2 mCi/ml [3H]-adenine in serum-free media (0.5 ml per well). The 3H-adenine was removed and each well was washed by the addition and removal of 1ml serum-free media. Serum-free media (1 ml) containing 100 mM 3-isobutyl-1-methylxanthine (IBMX) with or without the final required concentration of antagonist were added to each well and the cells were incubated for 30 min. Agonist (in 10 ml serum-free media) was added to each well and the plates were incubated for 30 min. When the intrinsic activity of the antagonists was assessed, the ligands were incubated for 5 h in the presence of 100 mM IBMX to maximize the changes in 3H-cAMP accumulation. The reaction was terminated by the addition of 50 ml concentrated HCl per well. The plates were then frozen, thawed and 3H-cAMP was separated from other 3H-nucleotides by sequential Dowex and alumina column chromatography.