P10 Rapid and Versatile MS-based immunoassays for Targeted Cytochrome P450 and Transporter Profiling in Tissue and Cell Culture Lysates

Frederik Weiss , SIGNATOPE GmbH, Reutlingen, Germany
Helen Hammer , SIGNATOPE GmbH, Reutlingen, Germany
Felix Schmidt , NMI Natural and Medical Sciences Institute at the University of Tuebingen, Reutlingen, Germany
Hannes Planatscher , SIGNATOPE GmbH, Reutlingen, Germany
Cornelia Sommersdorf , SIGNATOPE GmbH, Reutlingen, Germany
Thomas O. Joos , SIGNATOPE GmbH, Reutlingen, Germany
Oliver Poetz , SIGNATOPE GmbH, Reutlingen, Germany
In the late stage of pre-clinical drug development, the analysis of drug-drug interaction plays a pivotal role in detecting potential drug incompatibilities. The FDA currently recommends to measure mRNA as protein surrogate and functional tests for investigating the induction potential of a new chemical entity. During the last decade indirect quantification of proteins by measuring surrogate peptides in enzymatic digests of biological samples by mass spectrometry has become a widely used and accepted method. The end point analysis of CYPs, transporter, and UGT´s may allow a more accurate determination of the induction potential compared to mRNA and enzyme analysis. mRNA stability and regulation can lead to over and under-estimated induction effect. Enzyme activity measurements could be superimposed by simultaneous inactivation of the drug metabolizing enzymes by the drug itself. However, current proteomic analysis workflows for CYPs, transporter, & UGTs require large amount of sample material and a tedious membrane or microsome preparation step preventing the integration in routine processes.

Here, we present the development of mass spectrometry-based immunoassays for the analysis of phase I, II and III proteins. The method employs TXP-antibodies specific to short C-terminal peptide epitopes capable of enriching peptide groups. We generated antibodies targeting common epitopes present in so-called signature peptides derived from members of the cytochrome P450 family and drug transporters. These antibodies are applied in an immunoaffinity step prior quantification by high-resolution mass spectrometry (targeted selected ion monitoring). Thus, we are able to quantify 16 CYPs and 13 transporter directly from proteolytical digests of primary cells, tissue and tumors requiring only 5-50 µg protein extract. Due to low sample consumption, our immunoaffinity-coupled MS method enables the design of experiments in 96-well format, thereby reducing the required amount of human primary hepatocytes, which are the most precious part of an induction experiments. This semi-automated, highly parallel method has a throughput of 100s of samples per week and mass spectrometer. Using this method, we analyzed samples from pharmacokinetic studies in human hepatocytes addressing three different induction pathways. Additonally, we characterized the cytochrome P450- and transporter levels in liver tissue samples from healthy donors and tumor patients.