Pharmacodynamic assessments
Venous blood samples, drawn pre-dose and at regular time points after dosing, were diluted 1:1 with RPMI 1640 medium containing 25 mM Hepes and L-Glutamine (all BioWhittaker). The samples were incubated for 24 hours at 37°C (under 95% O2/5% CO2) with a fixed LPS concentration of 5 ng/mL. LPS-induced TNFα levels were measured in the supernantant using a solid-phase, two site chemiluminescent enzyme immunometric assay method (Immulite TNF-α) on a semi-automated analyzer (Immulite 1000, Siemens Medical Solutions Diagnostics B.V.). Blood samples collected before study drug administration were incubated with a concentration range of Org 48775-0 (0.01 to 60 μM) before start of the LPS incubation in to obtain anin vitro concentration response curve. LPS challenges were conducted by Good Biomarker Sciences (Leiden, The Netherlands)