Etanercept enbrel

SCW induced arthritic rats were randomly assigned to specific treatment groups and their baseline ankle diameter and withdrawal threshold values were recorded. Test article interventions were by oral gavage or subcutaneous injection either in a Prophylactic (P) or Therapeutic (T) regimen. In the prophylactic treatment regimen, compounds were administered once daily for 10 days starting on day 20 (1 day prior to SCW intravenous challenge) ending on day 29. The therapeutic treatment regimen entailed compound administration once daily for 8 days starting on day 22 (1 day post SCW intravenous challenge) ending on day 29. Non-arthritic and SCW control rats received vehicle (PEG 400:10% Tween 80 [1:9]) orally. Etanercept (Enbrel; Amgen Inc., Thousand Oaks, CA, USA) was purchased from Myoderm Limited, Norristown, PA, USA and was reconstituted in bacteriostatic water as per manufacturer’s instructions. Etanercept (subcutaneous (s.c.); 0.25 or 1 mg/kg/day) was administered either in prophylactic or therapeutic regimens. Rats in the dexamethasone group (Sigma Aldrich, St. Louis, MO, USA) received dexamethasone (per oral (p.o.); 0.3 mg/kg/day) suspended in (PEG 400:10% Tween 80 [1:9]). Buprenorphine (Sigma Aldrich) groups received Buprenorphine (p.o.; 0.05 mg/kg/day) suspended in saline in the therapeutic regimen. The dosing of compounds for all groups was stopped on day 29.

The culture medium was RPMI‐1640 supplemented with 2 mml‐glutamine, 1% sodium pyruvate, 50 μg/ml penicillin/streptomycin and 10% foetal calf serum (Invitrogen, Paisley, UK). Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of healthy volunteers using Lymphoprep (Axis‐Shield, Dundee, UK) and cultured with and without 10 µm zoledronate (Zometa; Novartis, Basel, Switzerland) for 16 hr; a combination of 10 ng/ml recombinant IFN‐γ and 20 ng/ml recombinant TNF‐α (both Miltenyi, Woking, UK) was used as positive control. For blocking experiments, anti‐IFN‐γ (B27; Biolegend, London, UK) and sTNFR p75‐IgG1 fusion protein (etanercept/Enbrel; Amgen, Cambridge, UK) were used at 10 μg/ml each. Cell culture supernatants were analysed in duplicate on a CLARIOstar microplate reader (BMG Labtech, Aylesbury, UK), using a sandwich ELISA kit for the detection of TNF‐α (Invitrogen).