Assay development considerations to improve drug tolerance in direct competitive ligand binding neutralizing antibody assays, pretreatment strategies.

Neutralizing anti-drug antibodies (ADAs) may affect safety, efficacy, and pharmacokinetic profile of a biotherapeutic drug and thus their assessment is of particular importance during immunogenicity testing. Neutralizing antibody (NAb) assays typically rely on NAbs ability to block the drug-target interaction. Higher NAb concentration and/or higher binding affinity of NAb to the drug, lowers the drug-target binding interaction. However, in the presence of high concentrations of residual circulating drug, as often seen for drugs with longer half-lives or in repeat-dose studies, NAbs may exist as drug bound complexes. In direct NAb assay formats, the NAb-drug complexes present in the sample could result in the NAb being unable to block the drug-target interaction eventually leading to a false negative response. The residual free circulating drug present in the sample may bind to the target in the NAb assay thereby competing with the drug used in the assay and inhibiting the assay signal, leading to a false positive response. For traditional ADA assays, multiple approaches involving acid treatment have been described to mitigate circulating drug interference issue. Here, we report two acid-treatment approaches that utilize the Dynabeads extraction with acid dissociation and Affinity Capture Elution (ACE) principle to improve drug tolerance in NAb assays.

Relative imbalance as etiology of laryngomalacia - A new theory.

Laryngomalacia literally means weak larynx. It is the most common cause of noisy breathing in infants and children constituting around 70% of cases. Its aetiology is not clear and various theories are proposed. Treatment remains following the child with regular weight monitoring in view of expected spontaneous resolution. However we cannot predict which child will resolve spontaneously and which child may need surgical intervention. We propose a new theory based on relative imbalance of demand supply of air, suggesting the increase in demand causing turbulent airflow, increasing suction pressure and causing collapse of laryngeal structures. This theory also helps us in predicting early, which child will resolve spontaneously and which child will need surgery. The methodology to evaluate hypothesis along with techniques and tools are also suggested.

Resolution of matrix interference: quantitative and quasi-quantitative ligand-binding assays case studies.

BACKGROUND: Matrix effects pose a constant challenge in developing robust ligand-binding assays to be validated for use in nonclinical and clinical study support. When notable matrix effects of any kind are present, it can render an otherwise sound method ineffective. We present two case studies detailing the mitigation of observed matrix effects. METHOD: A dimeric protein was removed from unknown samples in an anti-therapeutic antibody assay through protein extraction. Nonspecific matrix effects in a quantitative ligand-binding assays were mitigated through development of a specialized buffer. RESULTS: The protein extraction method reproducibly reduced the artificially high responses of naïve samples, enabling the accurate detection of anti-therapeutic antibodies. Design of experiments was used to evaluate and select the optimal components and associated concentrations in order to reduce the observed matrix effect to acceptable limits. CONCLUSION: Our results suggest there are multiple techniques available for the bioanalytical scientist to mitigate both matrix effects in ligand-binding assays.

Reversal of multidrug resistance by co-delivery of tariquidar (XR9576) and paclitaxel using long-circulating liposomes.

One of the major obstacles to the success of cancer chemotherapy is the multidrug resistance (MDR) often resulting due to the overexpression of drug efflux transporter pumps such as P-glycoprotein (P-gp). Highly efficacious third generation P-gp inhibitors, like tariquidar, have shown promising results in overcoming the MDR. However, P-gp is also expressed in normal tissues like blood brain barrier, gastrointestinal track, liver, spleen and kidney. To maximize the efficacy of P-gp inhibitor and reduce the systemic toxicity, it is important to limit the exposure of P-gp inhibitors and the anticancer drugs to normal tissues and increase their co-localization with tumor cells. In this study, we have investigated the co-delivery of the P-gp inhibitor, tariquidar, and cytotoxic drug, paclitaxel, into tumor cells to reverse the MDR using long-circulating liposomes. Tariquidar- and paclitaxel-loaded long-circulating liposomes showed significant resensitization of the resistant variant for paclitaxel, which could be correlated with an increased accumulation of paclitaxel in tumor cells. These results suggest that the co-delivery of the P-gp inhibitor, tariquidar, and the cytotoxicity inducer, paclitaxel, looks like a promising approach to overcome the MDR.