Safety, PK, and immunogenicity of i.d. and s.c. adalimumab administration
Adverse events were summarized by treatment group, in subsets of all treatment-emergent AEs, and separately for treatment-related AEs. Clinical laboratory and vital sign measurements were summarized by treatment and change from baseline was recorded. Summary statistics included number of subjects, mean, median, minimum, and maximum values (with standard deviation [SD]). Immunogenicity, i.e., anti-adalimumab antibodies, was reported descriptively.
For PK analyses, serum adalimumab concentrations were assessed in blood collected in 4 mL plain tubes (BD) after coagulation (30-60 minutes) and centrifugation (2000G for 10 minutes at 4°C), from day 1 (pre-dose) till day 71 post-dose. Adalimumab levels were quantified by fully automated ELISA as described (16) . Briefly, TNF was indirectly coated on microtitre plates. Serum was added and incubated. Immobilized adalimumab was subsequently detected using biotinylated rabbit anti-idiotype. The LOD for this assay is 10 ng/mL.
Anti-adalimumab antibodies were measured using a semi-quantitative radioimmunoassay as previously described (16). Briefly, samples were incubated with Sepharose-immobilized protein A (1.0 mg/test; Pharmacia Uppsala, Sweden) on its surface to capture IgG. After washing, radioactive iodine labelled F(ab’)2 fragments of adalimumab were added to detect drug-specific antibodies. The LOD for this assay 12 AU/mL.
Ex vivo whole blood challenge was performed to assess the effect of adalimumab on the release of cytokines by circulating immune cells and activation of these cells. Blood (6 mL) was collected in sodium heparin tubes (Becton Dickinson, NJ, USA) followed by stimulation with 2 ng/mL lipopolysaccharide (Sigma-Aldrich, Deisenhofen, Germany) and 25 µg/mL aluminium hydroxide (Alhydrogel 2%, Invivogen, Toulouse, France) for 24 hours at 37°C, 5% CO2. Culture supernatants were assayed for release of pro-inflammatory cytokines TNFα, IL-6, IL-1β, IFNγ and IL-8 using the Mesoscale Discovery multiplex immunoassay platform.