P311 Automated 96-Well Assay for the Assessment of Passive Permeability and P-gp Efflux During CNS Drug Discovery

Colin Lorenzten , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Shawn Gauby , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
David Nakamura , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Heather Zhang , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Erich Goldbach , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Shaila Hoque , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Bhushan Samant , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Pamela Santiago , Safety Evaluation, Elan Pharmaceuticals, South San Francisco, CA
Patrick Rudewicz , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
John-Michael Sauer , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
George Tonn , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
Kevin Quinn , Drug Metabolism and Pharmacokinetics, Elan Pharmaceuticals, South San Francisco, CA
The role of the ADME scientist in the drug discovery setting is to partner with discovery teams to influence the Structure-Property-Relationship towards compounds with tractable drug-like properties along with potency and selectivity.  For CNS drug targets, in addition to suitable solubility, metabolic stability and permeability, avoiding P-gp efflux liabilities are critical.  Cell based assays for determining permeability and P-gp efflux using traditional laboratory techniques is time consuming and laborious.   With that in mind, we have developed an automated screen to assess passive permeability and P-gp efflux in a 96-transwell format on a Microlab Star (Hamilton) liquid-handler coupled to a Cytomat cell culture incubator (Thermo).  We describe our assay workflow, which utilizes the liquid-handler to prepare dosing solutions and manipulate confluent monolayer cultures of MDR1 transfected MDCK–II cells (NKI) from pretreatment through to sample collection and processing.  Briefly, the passive permeability of each test-article (5 µM) is assessed in the forward (apical-to-basolateral) direction following pre-treatment of cells with a P-gp inhibitor (Tariquidar).  P-gp efflux is assessed by the reverse (basolateral-to-apical) over forward (apical-to-basolateral) ratio in the absence of inhibitor.   Controls are included to monitor tight-junctions (Atenolol), test-article recovery and assess boundary performance for permeability and P-gp efflux.  Test and control article concentrations are determined with a rapid LC-MS/MS assay following automated tuning (e.g. DQ2 software; Applied Biosystems).  Compounds are categorized to aid in the selection of lead series and the attrition during lead optimization.   Assay performance was verified using compounds of known permeability and P-gp liability.  In addition, in vitro data were compared to in vivo P-gp efflux determined from partition (Kp) ratios in the Mdr1 a/b (-/-) deficient mouse and wild type controls, with the assumption that efflux is approximately similar between human and mouse.  The assay was evaluated to test for the occurrence of false negatives and false positives to better understand the limitations of the assay.  With the proper use of controls to monitor for monolayer integrity and recovery, false positives were negligible.  False negatives were observed for highly permeable substrates (i.e. verapamil and quinidine) where efflux may be saturated at 5 µM.  In some cases this could be circumvented by testing these compounds at lower substrate concentrations.  Overall, a strong concordance was shown between our data, literature data and in vivo results.  This automated assay has enabled us to routinely and efficiently deliver high quality data to our discovery teams.   The rational use of these data by our teams has resulted in the advancement of various compound series, minimizing P-gp efflux liabilities and improving CNS penetration.