Automation of Cell-Based Drug Absorption Assays in 96-Well Format Using Permeable Support Systems

Abstract

Cell-based drug absorption assays, such as Caco-2 and MDCK-MDR1, are an essential component of lead compound ADME/Tox testing. The permeability and transport data they provide can determine whether a compound continues in the drug discovery process. Current methods typically incorporate 24-well microplates and are performed manually. Yet the need to generate absorption data earlier in the drug discovery process, on an increasing number of compounds, is driving the use of higher density plates. A simple, more efficient process that incorporates 96-well permeable supports and proper instrumentation in an automated process provides more reproducible data compared to manual methods. Here we demonstrate the ability to perform drug permeability and transport assays using Caco-2 or MDCKII-MDR1 cells. The assay procedure was automated in a 96-well format, including cell seeding, media and buffer exchanges, compound dispense, and sample removal using simple robotic instrumentation. Cell monolayer integrity was confirmed via transepithelial electrical resistance and Lucifer yellow measurements. Proper cell function was validated by analyzing apical-to-basolateral and basolateral-to-apical movement of rhodamine 123, a known P-glycoprotein substrate. Apparent permeability and efflux data demonstrate how the automated procedure provides a less variable method than manual processing, and delivers a more accurate assessment of a compound's absorption characteristics.

Expression of matrix metalloproteinases and tissue inhibitor of matrix metalloproteinases in the hair cycle

“…As a downstream cytokine regulated by the aforementioned signaling pathways, MMP-9 is an important collagenase involved in ECM remodeling ( 32 ). Previous studies suggested that MMP-9 serves an important role in the hair cycle ( 25 ). In the present study, the expression and activity of MMP-9 fluctuated, demonstrating peak activity during the catagen phase and minimum activity during the telogen phase in mice.…”

“…There are a few studies investigating the relationship between MMP-2, MMP-9, TIMP-1 and the hair cycle ( 25 ). MMP-2, MMP-9 and TIMP-1 have been suggested to be associated with the hair cycle in vitro and in vivo ( 26 ).…”

How to Determine the Role of the Microbiome in Drug Disposition

“…Cell-based drug absorption assays are among the most common method of in vitro screening of absorption parameters (Artursson et al, 2001) and are readily adopted for high-throughput screening (Larson et al, 2012). The Caco-2 cell line (ATCC HTB-37) is the preferred model for these types of assay because of its ability to differentiate into a monolayer resembling the enterocytes of the small intestines with characteristic expression of transporters including p-glycoprotein and OATP-A/B.…”

Complex High-Content Phenotypic Screening

“…Experimental test compounds are then added to this mock intestinal lining and permeability and transport characteristics of the compound are calculated. This component of Absorption, Distribution, Metabolism and Excretion (ADME)/tox (absorption, distribution, metabolism, and excretion) testing often determines whether a compound continues in the drug discovery process [59]. Development and implementation of a high density 384-well transwell plate may significantly expand phenotypic drug discovery for a range of different disease indications.…”