Comparison of analytical methods to detect instability of etanercept during thermal stress testing.

The aim was comparing the capability of a set of analytical methods to detect physical instability (focus on aggregation and structural changes) of etanercept during thermal stress testing as early as possible. Pre-filled syringes of Enbrel® 50mg from three batches were thermally stressed for one week at 50°C. Samples were taken at days 0, 1, 2, 3, 4 and 7, and analyzed with high-performance liquid size exclusion chromatography (HP-SEC), SDS-PAGE gel electrophoresis, dynamic light scattering (DLS), light obscuration, extrinsic fluorescence (Bis-ANS), far-UV circular dichroism (CD) spectroscopy, second derivative UV spectroscopy (UV), and enzyme-linked immunosorbent assay (ELISA). Thermal stress resulted in the formation of small soluble aggregates (HP-SEC, DLS) which were in part covalent (SDS-PAGE), and conformationally changed (Bis-ANS, CD, UV). No significant increase in subvisible particles was detected by light obscuration. An apparent increase in TNF-α binding to etancercept in the stressed formulations was found by ELISA. The three batches were comparable when unstressed, but showed slight differences in aggregation tendency. Bis-ANS fluorescence was most sensitive with respect to early-stage detection of heat-induced instability of etanercept (significant changes already at day 1), followed by HP-SEC (day 2) and DLS (day 3). This points towards a degradation mechanism involving exposure of hydrophobic patches due to partial unfolding followed by aggregation.

The presence or absence of antibodies to infliximab or adalimumab determines the outcome of switching to etanercept.

OBJECTIVE: The aim of this study was to test the hypothesis that the reason for non-response (caused by immunogenicity or not) to a first tumour necrosis factor (TNF) inhibitor defines whether a second TNF inhibitor will be effective. METHODS: This cohort study consisted of 292 consecutive patients with rheumatoid arthritis (RA), all treated with etanercept. A total of 89 patients (30%) were treated previously with infliximab or adalimumab ('switchers'), and the remaining 203 (70%) were anti-TNF naive. All switchers were divided into two groups: with and without antibodies against the previous biological. Differences in clinical response to etanercept between switchers with and without antibodies and patients who were anti-TNF naive were assessed after 28 weeks of treatment using changes in Disease Activity Score in 28 joints (DAS28). RESULTS: After 28 weeks of treatment, response to etanercept did not differ between patients who were anti-TNF naive and switchers with anti-drug antibodies (ΔDAS28=2.1 ± 1.3 vs ΔDAS28=2.0 ± 1.3; p = 0.743). In contrast, switchers without anti-drug antibodies had a diminished response to etanercept treatment compared to patients who were TNF naive (ΔDAS28 =1.2±1.3 vs ΔDAS28 = 2.1 ± 1.3; p = 0.001) and switchers with antibodies (ΔDAS28 =1.2±1.3 vs ΔDAS28 = 2.0 ± 1.3; p = 0.017). CONCLUSION: Patients with RA with an immunogenic response against a first TNF-blocking agent had a better clinical response to a subsequent TNF blocker compared to patients with RA without anti-drug antibodies. Hence, determining immunogenicity can be helpful in deciding in which patient switching could be beneficial and can be part of a personalised treatment regimen.

Decreased clinical response to infliximab in ankylosing spondylitis is correlated with anti-infliximab formation.

OBJECTIVES: Correlation of serum trough infliximab levels and antibodies to infliximab (anti-infliximab) with clinical response in ankylosing spondylitis. METHODS: In accordance with the international ASsessment in Ankylosing Spondylitis (ASAS) consensus statement, patients were treated with infliximab (5 mg/kg) every 6 weeks after a starting regimen. Preinfusion sera were collected at baseline, 24 and 54 weeks. At every visit, the 20% improvement response (ASAS-20) was assessed and laboratory tests performed. RESULTS: 24 of the 38 (63%) patients fulfilled ASAS-20 response criteria after 24 weeks of treatment and 21 (53%) after 54 weeks. After 54 weeks, 11 (29%) patients showed undetectable serum trough infliximab levels and detectable anti-infliximab; six of these patients developed an infusion reaction. Anti-infliximab was found significantly more often (p = 0.04) in ASAS-20 non-responders compared with responders at week 54. Serum trough infliximab levels were significantly (p<0.0001) lower in patients with (mean 0.02 mg/l) than in those without (12.7 mg/l) anti-infliximab. CONCLUSIONS: In ankylosing spondylitis, high levels of serum trough infliximab correlated with a good clinical response. Detection of anti-infliximab within 54 weeks is associated with undetectable serum trough infliximab levels, reduced response to treatment and increased risk of developing an infusion reaction.

Coughing in pre-school children in general practice: when are RAST's for inhalation allergy indicated?

To identify patterns of clinical history associated with extreme (high or low) probabilities of allergic sensitization in coughing children so as to restrict allergy testing to those with an intermediate probability of sensitization. A total of 752 children, aged 1-4, visiting their GPs for coughing (>or=5 days), were tested for IgE-antibodies to house dust mite, cat and dog [RadioAllergoSorbent Test (RAST)]. Parents completed a questionnaire on family history of atopy, breastfeeding, smoking, pets, and floor covering. Data of 640 children could be analyzed, 83 (13%) were IgE-positive. In a logistic regression analysis, a scoring formula for the prediction of being IgE-positive was constructed using variables from the patient's history. Significant contributors for sensitization were: age (3-4 yr), infantile eczema, positive family history of mite-allergy, sibling(s) with pollen-allergy, and smoking by parents. If only one of these characteristics is present, the probability of sensitization is < 25%. In such cases watchful waiting may be preferred over allergy testing. In other cases, a negative RAST may help to exclude sensitization, whereas a positive RAST helps to establish the diagnosis. Thus, acting on clinical history alone may save approximately 80% of RAST's. Patient history-derived information contributes to distinguishing children who are at low risk for sensitization to house dust mite, cat, and dog. The scoring formula may help GPs to identify children with a low probability of being sensitized. This may form the basis for watchful waiting. In others, allergy testing may be useful to gain more diagnostic certainty.