Hepatobiliary Dispostion and Drug Transport
Prediction of the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of compounds in pharmaceutical development are essential aspects of the drug discovery process. B-CLEAR® (sandwich-cultured hepatocytes) is an in vitro system that is used to analyze and predict the in vivo hepatobiliary disposition (hepatic uptake, biliary excretion, and biliary clearance) and transporter-based hepatic drug-drug interactions. When sandwich-cultured hepatocytes are exposed to calcium, junctions remain tight and compound may be retained in both cell and bile. If calcium is not present, junctions open and the contents of the bile pockets are released into the media. Measuring the difference of between these two conditions provides information on uptake and excretion properties of test compound. Currently, uptake experiments are performed manually in 6- and 24- well formats. Automation of 6- and 12- well formats had not been successful due to complexities surrounding experimental constraints associated with timing, temperature, and washing procedures. A 24-well format has lower volume requirements and is higher density, enabling automation of uptake studies in this format. In this poster, we have compared results from automated 24-well format with manual 24-well to assess experimental performance. The automated 24- well uptake may offer a more transferable format which is equally robust and less labor intensive.
- Automation of the B-CLEAR® technology in a 24-well format is feasible and offers an equally robust assay.
- Differences between the automated and manual formats (total accumulation and NSB) have minimal effect on results.
- Future experiments will involve optimization of wash and more extensive compound testing and variability analysis.
- To meet assay constraints, current throughput is 48 wells per 40 minutes
- NSB values are less in manual studies indicating a more effective final wash.
- Accumulationin manual versus automated uptake studies are comparable, althoughthere is noticeable difference between formats (slightly less inautomated; degree of difference is compound dependent)
- Whenaccumulation is corrected for NSB (accumulation – NSB), the ratiobetween cells and cells + bile for automated and manual studies areequivalent
- NSB (residual compound) decreases with increasingwash volume and number of washes. Mixing (aspirate – dispense) alsodecreases NSB, but may disrupt the cell layer.
- Execution time of final wash must be limited in consideration of
- Biology requirements (exposure of cells to wash buffer is limited to 20 seconds) and to increase throughput.
- Further wash studies will be done to optimize most effective wash in least amount of time.
- Observationof lower accumulation value from automated uptake studies cannot beattributed to incubation on a 37oC surface instead of a 37oC incubator