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)