Thursday, March 18, 2010

Hepatocyte Assays in 384-Well Microtiter Plates

Cytochrome P450 enzymes are key players in the metabolism of drugs within the body. Therefore, it is essential to understand how these enzymes can be affected by xenobiotics with regards to inhibition, induction, and toxicity to avoid potential drug-drug interactions. Typically this information has been gathered by combining the data from multiple experiments. This process is time-consuming and labor intensive. And it may lead to misinterpretation when combining the data due to the variability in each assay. A multiplexed format provides a method to obtain multiple readouts from a single microplate well, which may attenuate these concerns. Here we show the ability to monitor CYP1A and -3A induction, combined with cytotoxicity measurements, from a single well using cryopreserved hepatocytes. The assay utilizes BioTek’s EL406 Combination Washer Dispenser, Precision Microplate Pipetting System, and Synergy H4 Multimode Microplate Reader.

The assay is carried out over a five day process. Cryopreserved hepatocytes are thawed, diluted, and plated into Collagen I coated microtiter plates. After a media exchange on day two, the cells are incubated over a 48-hour period with a titration of various compounds. Following the completion of the incubation period, two separate substrates are added to the cells to monitor the induction of the cytochrome P450 enzymes. Luciferin-IPA from Promega Corporation is used in conjunction with CYP3A, and Resorufin Ethyl Ether is used with CYP1A. Luciferin-IPA is cleaved by the CYP3A enzyme present in the well to create luciferin. The luciferin then reacts with luciferase and ATP from the Luciferin Detection Reagent to create a stable luminescent signal. The Resorufin Ethyl Ether is also converted to Resorufin by CYP1A, and fluorescence is then detected upon excitation. Finally, CellTiter-Glo is added to measure cell viability in each well. Figure 1 explains the procedure in more detail.

Due to the fact that primary hepatocytes are incorporated, coupled with the multi-day assay procedure, sample sterility is essential during the entire process. This was accomplished by placing the two instruments used for aspiration and dispensing, the EL406 and Precision, each into standard biosafety cabinets.

Data was analyzed in order to determine fold induction of the two cytochrome P450 enzymes and cell viability from each concentration of compound. By using this type of analysis (see Figure 2), it is easy to see the effect that the compound has on the two CYP enzymes. Rifampicin, a well known CYP3A inducer shows high fold induction of the enzyme across a wide range of concentrations, but does not show any effect on CYP1A, as expected.

Figure 3 further demonstrates how the cell viability measurement can be used to help explain the changes in fold induction that are seen across each compound titration. Lansoprazole induces CYP1A at lower concentrations of the compound, but then this effect decreases dramatically above 50 ┬ÁM. By including the viability measurement, it becomes obvious that this decrease is due to cytotoxicity from the compound at higher concentrations.

The data and process explained here demonstrate how automated, multiplexed cell-based CYP assays can simplify workload, save time and effort, and generate the data needed from today’s ADME/Tox laboratory.

What ADME/Tox assays would you like to see multiplexed? How can BioTek simplify the workload and processes that are currently in place in your lab?

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