Automation of Pre-coated PAMPA Plates Improves Predictability ...

ResultsThe Corning® Gentest Pre-coated PAMPA Plate System simplifiesthe PAMPA process since effort is not required to form andprepare the membrane prior to adding the compounds. Figure 1compares the traditional PAMPA process flowchart and the newPAMPA process flowchart. The traditional process requires theuser to perform coating of the filter plate before setting up theassay. In the new process, the filter plate is ready to use, thereforethe assay can be started by the addition of compounds tothe Donor wells, addition of buffer to the Acceptor wells, andcoupling the Donor and Acceptor plates together for incubation.Overall time required to run the assay is greatly reduced whencompared to traditional methods.The new artificial membrane is designed not only to improvethe stability, but also to improve the PAMPA model. TraditionalPAMPA membrane has incorrect predictions for some high permeabilitycompounds, presumably due to the excessive amount of solventspresent in the membrane structure. Figure 2 compares thestructure of the artificial membrane of the traditional PAMPA andthe Corning Gentest Pre-coated PAMPA Plate System. The traditionalPAMPA membrane is prepared by adding a solution of lipids.As a result, the porous filter is soaked with the solvent of thelipids, creating a long permeation pathway for the compounds.In comparison, the Corning Gentest Pre-coated PAMPA PlateSystem PAMPA membrane is prepared by constructing thin layersof lipids inside the porous filter, using volatile solvents whichevaporate after coating. Therefore, the permeation pathway ismuch shorter and is a better mimic of the biological membrane.The Corning Gentest Pre-coated PAMPA Plate System is fullyautomation compatible. This is demonstrated by carrying outthe entire assay process using a Tecan® Genesis 150 liquidhandler which is integrated to a plate reader using a robot arm.The automation deck layout is shown in Figure 3 and examplesof robotic handling are shown in Figure 4.To validate the stability of the pre-coated plates, experimentshave been carried out using plates coated at different times.Figure 5 compares the results obtained from three plates coatedat different times and used for assay on the same day. The resultsobtained from the one-year-old plate and the six-month-old plateare almost identical to the results obtained from a freshly coatedplate. These results indicate that the Corning Gentest Pre-coatedPAMPA Plate System is stable for at least one year when stored at-20°C and is highly reproducible from plate to plate.For Research Use Only. Not for use in diagnostic or therapeutic procedures.For a listing of trademarks, visit us at www.corning.com/lifesciences/trademarks.Other trademarks are the property of their respective owners.Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-0001To validate the automation of the pre-coated plates, theresults obtained using automated assays were compared tothe results obtained using manual assays. The results shown inFigure 6 confirmed that both methods yielded the same results,while the automated methods generated data with tighterCVs, presumably due to more accurate pipetting compared tomanual pipetting.Figure 7 compares the performance of a traditional PAMPA andthe Corning Gentest Pre-Coated PAMPA Plate System by analyzingthe correlation of the permeability data with the humanabsorption data of 35 compounds. Using the traditional PAMPA,there is a group of compounds with high human absorption propertiesthat are under-predicted (false negative). This group ofcompounds are correctly predicted using the Corning GentestPre-Coated PAMPA Plate System.ConclusionsThe Corning Gentest Pre-Coated PAMPA Plate System is fully automationcompatible and improves the PAMPA model through thefollowing characteristics:w Pre-coated plates improve process efficiency because themembrane preparation step is skippedw Pre-coated plates are stable and highly reproducible fromplate to platew The new artificial membrane better mimics the biological membraneand improves PAMPA predictability, as demonstrated bythe improved correlation with human absorption dataReferences1. Kansy, M., Senner, F., Gubernator, K. J. Med. Chem. 41:1007(1998).2. Avdeef, A., Strafford, M., Block, E., Balogh, M., Chambliss, W.,Khan, I. Eur. J. Pharm. Sci. 14:271 (2001).3. Ruell, J.A., Avdeef, A., Du, C., Tsinman, K. A Simple PAMPAFilter for Passively Absorbed Compounds, Poster, ACS NationalMeeting, Boston, August 2002.4. Chen, X., Murawski, A, Patel, K., Crespi, C. L., Balimane, P.,Pharmaceutical Research, in press (2008).Corning acquired the Falcon® and Gentest brands.For additional Corning product, technical, or distributor information,please e-mail us at CLSTechServ@corning.com, visit ourwebsite www.corning.com/lifesciences or call 800.492.1110.Outside the United States, call 978.442.2200. For informationon the acquisition, visit www.corning.com/discoverylabware.Corning IncorporatedLife Sciences836 North St.Building 300, Suite 3401Tewksbury, MA 01876t 800.492.1110t 978.442.2200f 978.442.2476www.corning.com/lifesciences© 2012, 2013 Corning Incorporated Printed in U.S.A. 5/13 CLS-DL-GT-053