In vitro activity: VUF10460 is a non-imidazole agonist of the histamine H4 receptor which binds to rat H4 receptor with a pKi of 7.46. Radioligand binding studies showed that VUF10460 displayed approximately a 50-fold selectivity for the rat H4 receptor over the H3 receptor, VUF10460, possibly due to the lack of imidazole heterocycle. In conscious rats, immethridine and methimepip significantly reduced (66% and 48% inhibition, respectively) the gastric lesions induced by HCl; HCl-induced lesions were also significantly enhanced by the H4 receptor agonists VUF10460 and VUF8430, suggesting the activation of histamine H4 receptors by the selective agonists VUF10460 and VUF8430 aggravated the ulcerogenic effects of 0.6 N HCl.

Kinase Assay: VUF10460 binds to rat H3 and H4 receptor with pKi values of 5.75, and 7.46, respectively. VUF10460 displays approximately a 50-fold selectivity for the rat H4 receptor over the H3 receptor. Displacement radioligand binding assay was performed in 50 mM Tris–HCl buffer (pH 7.4 at 25 °C) containing homogenized rat histamine H3 receptor-transfected cells, with or without competing ligands (10– 4 to 10– 11 M), in the presence of 1 nM [3H]Nα-methylhistamine (85 Ci/mmol; Perkin Elmer, USA) in a total volume of 200 μl. The binding reaction was incubated for 60 min at 25 °C, and terminated by rapid filtration over Unifilter GF/C 96-well filterplates (PerkinElmer, USA) pretreated with 0.3% 750 kDa polyethylenimine, followed by three washes with ice cold 50 mM Tris–HCl (pH 7.4 at 4 °C). Radioactivity retained on the filter was determined by liquid scintillation counting on the Microbeta Trilux with 25 μl Microscint“O”(TM)

Cell Assay: HEK 293 T cells were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 50 IU/ml penicillin, and 50 μg/ml streptomycin in 5% CO2 humidified atmosphere at 37 °C. Approximately 4 million cells were seeded in a 10-cm dish and cultured overnight before transfection. For transfection of each dish of cells, the transfection mixture was prepared in 0.5 ml serum-free DMEM and contained 5 μg of rat histamine H4 or H3 receptor plasmid and 15 μl of 1 mg/ml 25 kDa linear polyethyleneimine (Polyscience, Inc., USA) and vortexed immediately. The mixture was incubated for 10–15 min at room temperature before it was added into the monoloyer cell culture loaded with 6 ml fresh cell culture medium. Two days after transfection the cells were scraped, collected as pellet by centifugation and stored at – 20 °C until use.