Etanercept (Enbrel®) ELISA Kit

Enzyme-linked immunosorbent assay (ELISA) for the quantitative determination of free Etanercept (Enbrel®) in serum and plasma. The Assay Genie Etanercept (Enbrel®) ELISA Kit has been especially developed for the quantitative analysis of free etanercept in serum and plasma samples and is for research use only.

Solid phase enzyme-linked immunosorbent assay (ELISA) based on the sandwich principle. Standards and samples (serum or plasma) are incubated in the microtitre plate coated with the reactant specific for free etanercept (Enbrel®). Following incubation wells are washed and then horse radish peroxidase (HRP) is added and binds to Etanercept. After incubation, the wells are washed, and the bound enzymatic activity is detected by addition of chromogen-substrate. The colour developed is proportional to the amount of free etanercept (Enbrel®) in the sample or standard. Results of samples can be determined directly using the standard curve.

Etanercept (Enbrel®) is a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75) tumour necrosis factor receptor (TNFR) linked to the Fc portion of human IgG1. The Fc component of etanercept contains the CH2 domain, the CH3 domain and hinge region, but not the CH1 domain of IgG1. Etanercept consists of 934 amino acids and has an apparent molecular weight of approximately 150 kilodaltons.

Etanercept binds specifically to tumour necrosis factor (TNF) and blocks its interaction with cell surface TNF receptors. Elevated levels of TNF are found in involved tissues and fluids of patients with rheumatoid arthritis (RA), psoriatic arthritis, ankylosing spondylitis (AS), and plaque psoriasis. Two distinct receptors for TNF (TNFRs), a 55 kilodalton protein (p55) and a 75 kilodalton protein (p75), exist naturally as monomeric molecules on cell surfaces and in soluble forms. Biological activity of TNF is dependent upon binding to either cell surface TNFR. Etanercept is a dimeric soluble form of the p75 TNF receptor that can bind to two TNF molecules. Etanercept inhibits binding of both TNF alpha and TNF beta (lymphotoxin alpha [LT alpha]) to cell surface TNFRs, rendering TNF biologically inactive.

Etanercept is a tumor necrosis factor (TNF) inhibitor used to treat various autoimmune conditions including certain types of arthritis; rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis. Etanercept is also used to treat particular skin conditions such as plaque psoriasis.

After administration of 25 mg of ENBREL® by a single subcutaneous (SC) injection to 25 patients with RA, a mean ± standard deviation half-life of 102 ± 30 hours was observed with a clearance of 160 ± 80 mL/hr. A maximum serum concentration (Cmax) of 1.1 ± 0.6 mcg/mL and time to Cmax of 69 ± 34 hours was observed in these patients following a single 25 mg dose. After 6 months of twice weekly 25 mg doses in these same RA patients, the mean Cmax was 2.4 ± 1.0 mcg/mL (N =23). Patients exhibited a two- to sevenfold increase in peak serum concentrations and approximately four-fold increase in AUC0-72 hr (range 1 to 17-fold) with repeated dosing. The pharmacokinetic parameters in patients with plaque psoriasis were similar to those seen in patients with RA.

In another study, serum concentration profiles at steady state were comparable among patients with RA treated with 50 mg ENBREL® once weekly and those treated with 25 mg ENBREL® twice weekly. The mean (± standard deviation) Cmax, Cmin, and partial AUC were 2.4 ± 1.5 mcg/mL, 1.2 ± 0.7 mcg/mL, and 297 ± 166 mcg·h/ mL, respectively, for patients treated with 50 mg ENBREL® once weekly (N = 21); and 2.6 ± 1.2 mcg/mL, 1.4 ± 0.7 mcg/mL, and 316 ± 135 mcg·h/mL for patients treated with 25 mg ENBREL® twice weekly (N = 16). No formal pharmacokinetic studies have been conducted to examine the effects of renal or hepatic impairment on ENBREL® disposition. Patients with juvenile idiopathic arthritis (JIA) (ages 4 to 17 years) were administered 0.4 mg/kg of ENBREL® twice weekly for up to 18 weeks. The mean serum concentration after repeated SC dosing was 2.1 mcg/mL, with a range of 0.7 to 4.3 mcg/mL.

Limited data suggests that the clearance of ENBREL® is reduced slightly in children ages 4 to 8 years. Population pharmacokinetic analyses predict that administration of 0.8 mg/kg of ENBREL® once weekly will result in Cmax 11% higher, and Cmin 20% lower at steady state as compared to administration of 0.4 mg/ kg of ENBREL® twice weekly. The predicted pharmacokinetic differences between the regimens in JIA patients are of the same magnitude as the differences observed between twice weekly and weekly regimens in adult RA patients. Patients treated with ENBREL® are at increased risk for developing serious infections that may lead to hospitalization or death. Patients should be closely monitored for the development of signs and symptoms of infection during and after treatment with ENBREL®, including the possible development of tuberculosis in patients who tested negative for latent tuberculosis infection prior to initiating therapy.

It has been reported that patients receiving etanercept may develop antibodies that interfere with monoclonal antibody laboratory assays. Serum concentration of ENBREL® might be related to predict some clinical outcome during maintenance therapy. It was also possible that the surveillance of circulating ENBREL® concentration during maintenance therapy represents a direct and/or indirect factor for immunogenicity and some other side effects. In this context, identification of biomarkers for (non-)response and risk factors for adverse drug reactions that might be related to serum concentrations and maintaining the effective minimum concentration of ENBREL® in order to potentially avoid some side effects with a reliable method might be beneficial.