Abstract B139: Determination of the plasma inhibitory activity of drugs using cellular kinase assays

Abstract

Abstract Anticipation of the in vivo activity of new investigational drugs is challenging. In the context of the living body, compounds are subjected to degradation, modification or binding to plasma proteins - mechanisms that may compromise the activity predicted from target-specific in vitro assays. As such assays are performed in artificial cell-culture media it was our aim to measure the activity directly in human plasma, as initially described by Levis et al.1. We hence established a method to analyze the activity of kinase inhibitors in human or rodent plasma (plasma inhibitory activity, PIA) in high throughput compatible ELISA-based cellular kinase assays for e.g. FLT-3 or BCR-Abl. Testing reference compounds such as Sunitinib or Sorafenib, we could confirm the expected correlation between the plasma binding of a compound and the loss of its activity in this assay setup compared to the activity in serum free medium. Furthermore we observed a donor dependent interference of the plasma with assay performance. This issue was solved by appropriate plasma heat-inactivation which made confirmation of compound stability to heat treatment mandatory before generating pharmacokinetic data using this methodology. Our aim is to establish the PIA assay for our panel of more than 30 kinases and analyze the activity of important reference kinase inhibitors spiked in human plasma. In the next step samples from mice treated with reference compounds shall be tested. This way, we want to compile reliable in vitro PIA assays to better predict the in vivo effect of kinase-directed drugs and to determine the amount of freely available active test substance in the blood of treated patients. 1 = Levis et al.; Blood, 2006; 108:3477 Citation Format: Kira E. Boehmer, Holger Weber, Daniel Feger, Marianne Birkle, Oliver Siedentopf, Melanie Mueller, Sarah Umber, Jan E. Ehlert. Determination of the plasma inhibitory activity of drugs using cellular kinase assays. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B139.