Fully Automated 24-well Madin-Darby Canine Kidney (MDCK) Permeability Assay Utilizing the Hamilton Microlab STAR Liquid Handling Platform

Abstract:
Permeability assays are used to elucidate the structure-activity relationships in the hit-to-lead stage, rank order compounds for in vivo pharmacokinetics studies, and predict the potential for oral absorption. It is important to have reliability, accuracy and precision for such assays especially when used in mid-high throughput screening. This application note demonstrates the fully automated 24-well Madin-Darby Canine Kidney (MDCK) permeability assay using Hamilton Microlab STAR liquid handling workstation. 


Introduction:

A robust permeability assay is an essential tool in the drug discovery process. Mandin Darby Canine Kidney (MDCK) cells are a common model for studying drug transport mechanisms in distal renal epithelia. Like Caco-2 cells, MDCK cells differentiate into columnar epithelium and form tight junctions when cultured on semi-permeable membranes. Primarily for assessing passively absorbed compounds, drug permeability data from MDCK cell assays have been shown to be similar to permeability data from Caco-2 cell assays.1 Permeability data is also an important input parameter for in silico software programs which can be used to model oral bioavailability and other ADME (Absorption Distribution Metabolism Excretion) parameters. MDCK cells grown to confluence on permeable membranes and are widely used to examine passive permeability and active transport. These assays have typically been run manually or have been partially automated requiring an operator’s presence at all times. We have successfully developed a fully automated assay utilizing a Hamilton Microlab STAR integrated with select third party peripheral devices to suit the assay requirements.

The method was validated using 23 marketed drugs with diverse physico-chemical properties and a wide range of published apparent permeabilities (Papp). The validation set also included subsets of drugs required for GastroPlus and SimCyp ADME simulation software allowing the simultaneous validation of the method while providing required data for in silico modeling. The automated method was also tested against a 12-well manual method using 13 marketed drugs and eight Genentech compounds to ensure the rank order and low, moderate, and high permeability classifications were similar between the different methods. Hamilton Robotics provides world class automation solutions for a variety of applications with increased robustness, flexibility, precision and accuracy using its unique CO-RE technology. This application note demonstrates the automation capabilities of Hamilton Microlab STAR and how it can be used to fully automate the MDCK permeability assay quickly and reliably.

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Application note

 
 
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