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May 15, 2011 (Vol. 31, No. 10)

Human Hepatocytes in HTS DDI Studies

Technique Addresses Inhibition and Induction to Improve Screening for Drug Safety

  • Advancement of techniques to achieve efficient and relevant outcomes occurs when technologies merge at critical moments. In the pharmaceutical industry, techniques such as in silico drug design and microarray analysis illustrate the power of combined technologies to transform the manner and expectations of how pharmaceutical research is performed. The refinements allow for not only greater generation of data but also deeper understanding of that which is observed.

    Drug safety advances have also been notable. The combination of high-throughput screening (HTS) technology, development of substrate probes, and quality of relevant cellular systems has led to the meaningful development of techniques for the assessment of drug-drug interactions (DDI). Traditionally, DDI occur when one drug affects a second drug’s efficacy or toxicity due to perturbing the metabolic capacity of a patient by the inhibition and induction of cytochrome P450 (CYP) enzymes.

  • Inhibition

    Click Image To Enlarge +
    Table. IC50 values generated from cryopreserved pooled human hepatocytes and human liver microsomes using P450-Glo substrates for CYP1A2, -2C9, and -3A4.

    Inhibition of CYP enzymes diminishes the metabolic activity of an enzyme, resulting in accumulation of the drug that reduces therapeutic efficacy or increases toxicity. For example, bergamottin, a component of grapefruit juice, inhibits CYP3A4, the enzyme responsible for the metabolism of more drugs than any other enzyme. Clinically significant DDI have been associated with consumption of grapefruit juice and numerous drugs that are metabolized by CYP3A4 such as midazolam and atrovastin.

    To test for this phenomenon in vitro, microsomes, the subcellular fraction containing CYP enzymes, have been used as the gold standard. However, results from microsomal inhibition may not correlate to in vivo responses due to other cellular events. The use of hepatocytes in lieu of microsomes leads to better in vivo correlation of clinically relevant inhibition when intracellular concentrations of a drug are affected by drug transporters or other events.

    However, the use of hepatocytes to augment or potentially replace microsomes as the primary system for inhibition studies requires these cells to emulate key microsomal attributes while increasing the quality of data due to whole cell architecture.

    To address these needs, cryopreserved pooled human hepatocytes have become readily available to the research community from companies such as Celsis, providing an average of responses from multiple donors to mitigate idiosyncratic responses and offering consistency among lots to minimize revalidation of reagents.

    This mimics the power of averaging CYP enzymatic activities found in human liver microsomes and, in addition, includes the full complement of Phase I and Phase II drug metabolism, uptake transporter activities, and cell membrane barrier.

    The utility of human hepatocytes to determine inhibition has been demonstrated in recent studies by comparing the inhibition of four clinical drugs with known transporter-enzyme interplay. In one study, the authors concluded that determination of inhibition and enzymatic activities is not intuitive between microsomal and hepatocytic systems, and highlighted potential shortcomings when using microsomes to predict clinical responses.

    Promega, BioTek Instruments, and Celsis collaborated on research that was presented in a poster at “LabAuto” earlier this year. The poster described the use of pooled human hepatocytes in HTS format to measure inhibition of CYP enzymes. The authors developed a high-quality 384-well system for dispensing and incubating pooled human hepatocytes, as well as measuring hepatocytic responses using luminogenic specific substrates.

    The results of the inhibition using pooled human hepatocytes were compared to human liver microsomes under the same conditions. The derived inhibition constant Ki was similar for most of the test compounds except for a few with known transporter interactions like verapamil (Table).

    Pharmaceutical researchers now have a new technique to further study inhibition in a more relevant manner, while retaining the work flow expected with microsomal inhibition assays.

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