1. In Vitro Sciences Abstract The extent to which a drug binds to plasma proteins is a commonly derived parameter during drug discovery and development. It is assumed that only free, unbound, systemic drug is available to cause an efficacious effect in vivo, although this view has recently been challenged [1]. Nevertheless, plasma protein binding remains an integral parameter to assess to aid in the evaluation of pharmacological, pharmacokinetic and toxicological data. In common with other areas of in vitro science, recent product development in this area has been extensive and there are now different apparatus available for use to assess plasma protein binding in the modern DMPK laboratory. To assess the applicability of some of these new apparatus, we have compared the human plasma protein binding of atenolol (low plasma protein binding), testosterone (medium plasma protein binding) and warfarin (high plasma protein binding) using recently launched apparatus. These include: Fast-Micro-equilibrium DIALYZER™ devices, DispoEquilibrium DIALYZER™ devices, RED™ devices and the HTDialysis 96™ well plate and we have compared the binding values obtained with more traditionally encountered techniques such as equilibrium dialysis using Dianorm™ dialysis cells, ultrafiltration (Centrifree™ devices) and ultracentrifugation. This work has been conducted as part of an ongoing validation plan within Quotient Bioresearch and it is hoped the outcomes will aid in overcoming some of the commonly encountered issues that are associated with assessing plasma protein binding in vitro, namely, low throughput, problematic compounds (typically highly lipophilic compounds) and low sample volumes (typically from neonatal plasma). [1] The effect of plasma protein binding on in vivo efficacy: misconceptions in drug discovery, Dennis A. Smith, Li Di and Edward H. Kerns, Nature Reviews: Drug Discovery, 9 December 2010, 929 - 939. Data Processing Atenolol, testosterone and warfarin were spiked into human plasma at 10 µg/mL. All assessment of plasma protein binding was performed in triplicate at 37ºC as below. Methods Results As anticipated, for our highly bound drug, warfarin, there was good agreement between most of the techniques used (range 98.40 – 98.47% bound). Binding was slightly higher with RED™ devices (98.68%) and highest using DispoEquilibrium DIALYZER™ devices (99.25%). With testosterone, which is less bound than warfarin, greater variation was observed across the apparatus used. There was excellent agreement between ultrafiltration and ultracentrifugation (93.05 and 93.14%, respectively). Slightly higher binding values were obtained using the other devices (range 94.16 – 95.02%). Similar to warfarin, the highest binding value was observed using DispoEquilibrium DIALYZER™ devices (96.51%). With atenolol, where plasma protein binding is low, there was greatest variation with binding ranging from 0% bound using the traditional Dianorm™ dialysis cells, up to a maximum of 35.46% bound using the DispoEquilibrium DIALYZER™ devices. Results Kathryn Sweeney, Vicky Birks, Stephanie Geoffroy, Richard Cole and Guy Webber. Quotient Bioresearch Ltd. Northamptonshire, UK. T +44 (0) 1933 319900 E metabolism@quotientbioresearch.com www.quotientbioresearch.cometabolism@quotientbioresearch.comISSX 17 th North American Regional Meeting 16-20 October 2011 Samples of plasma, dialysate, ultrafiltrate and supernatant were analysed by UPLC-MS/MS (Acquity-TQD, Waters Corp TM ).. For the technique of equilibrium dialysis the extent of protein binding of test compound was calculated from the following expression using concentrations at the end of the dialysis period: For the techniques of ultrafiltration and ultracentrifugation the extent of protein binding of test compound was calculated from the expression: Plasma Protein Binding: A Comparison of Techniques to Help Overcome Common Issues Encountered During In Vitro Testing For reprints or further information please contact: Guy.webber@quotientbioresearch.com