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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

  • Induction

    Click Image To Enlarge +
    Figure. Induction responses for CYP1A2 and -3A4 as measured by metabolism of ethoxyresorufin and P450-Glo™ 3A4 IPA substrates, respectively, after treatment with rifampicin (A) and lansoprazole (B). The fold induction was derived by dividing the metabolic activity of the treated hepatocytes by the metabolic activity of the untreated hepatocytes. Viability was measured by ATP content using CellTiter-Glo.

    Induction of CYP enzymes increases the metabolism of a drug, resulting in a reduction of efficacy of the intended therapy. For example, certain antibiotics like rifampicin can increase the metabolism of birth control drugs, rendering them ineffective.

    As with inhibition DDI, induction potential must be assessed prior to exposure in patients. Human hepatocytes are the gold standard for determining induction potential. However, the commonly used 24-well format limits the number of wells per plate and the cost per well. Therefore, a migration to 96-well and 384-well induction models would offer a higher capacity to generate the required data with the same resources.

    Though hepatocytes in 96-well format have been shown to be an appropriate model for induction, the 384-well format has not been reported in the literature. A second poster, presented by the group at “ISSX”, described validation of a 384-well format. The 96- and 384-well formats were compared to ensure that miniaturization of the system did not impact the function of the hepatocytes or the derived values. To increase the quality of the data, a triplex system was developed to measure CYP1A and -3A4 activities along with viability from a single well.

    In doing so, the efficient use of hepatocytes and dosing solutions reduced potential variability from inter-well and inter-plate cell culturing and multiple drug-dispensing steps. That is, from a single dispensed dose, functional data was obtained for two induction endpoints and a viability endpoint. The CYP1A family was measured by the fluorescent substrate ethoxyresorufin, CYP3A4 by the luminogenic substrate P450 Glo™ CYP3A4 IPA (Promega), and viability by the luminogenic assay CellTiter-Glo®.

    The authors presented a 384-well system with robustness and consistency that meet HTS criteria while retaining the induction mechanism of human hepatocytes. In addition, the viability assessment allowed for normalization of the data as well as the potential to explain differences in the EC50 curves.

    For example, rifampicin showed lower induction at the high end of the curve while the viability remained constant, which may be interpreted as a suppression of the induction process or the inhibition of CYP3A4 enzyme (Figure, A).

    In contrast, lansoprazole showed a lower induction at the higher doses along with lower viability, allowing for the interpretation that toxicity of lansoprazole was responsible for the lower induction (Figure, B).

    The combination of an HTS platform and triplex methodology with human hepatocytes creates a powerful new technique for pharmaceutical scientists to assess induction potential. In addition, triplex methodology provides for efficient use of resources such as hepatocytes and dosing solutions, for reducing variation in responses compared to combining separate data points, and for normalization of data by viability parameter. The latter provides a critical value to explain a deviation from a sigmoidal response curve due to cellular toxicity or other cellular events.

  • Integration

    The responsibilities of pharmaceutical scientists have increased to meet a variety of challenges. The pharmaceutical industry is being pressured by regulatory agencies to provide extensive preclinical data to assess drug safety issues, such as DDI, before embarking on clinical development of new drugs. In addition, pharmaceutical scientists have internal pressure to screen more compounds with limited resources while producing clinically relevant and actionable information.

    New technologies and integrated platforms must be developed to meet these evolving needs. Two such platforms, HTS induction and inhibition systems using human hepatocytes, offer the potential to meet the challenges of today’s drug discovery programs by merging formerly separate technologies and reagents in order to provide new and potent techniques.



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