Use of Cold Compounds and High Resolution Mass Spectrometry to Quickly Characterize Drug Uptake in Cryopreserved Human Hepatocytes

GOAL Using cold compounds, we wanted to present a compelling set of data to show that hepatocytes and HRAM spectrometry can be used to obtain at least screening-quality information with regard to whether a test article is a substrate of the solute carrier uptake transporters. METHODS In this study, we assessed the use of cryopreserved hepatocytes for evaluating whether test articles are substrates of one of the OATPs with the oil filtration method [Parker and Houston, 2008]. Hepatocytes suspended in incubation media containing a test article for up to 3 minutes at both 4oC and 37oC were filtered through a layer of silicone oil using centrifugation. The cells in the receiving layer containing 2 N NaOH were disrupted and the clarified solutions were analyzed by an Acquity UPLC coupled to a Waters Q-Tof Premier. Method development for the test articles was performed in parallel with a generic gradient and no manual tuning (< 1 day). Several test articles were screened for accumulation in the hepatocytes. Also, several compounds were screened for their ability to reduce the uptake of typical probe substrates of OATP1B1, OATP1B3 and OATP2B1, estradiol-17s-glucuronide and estrone-3-sulfate. CONCLUSIONS The use of cryopreserved hepatocytes and high resolution mass spectrometry proved to be an effective combination for characterizing uptake transporters without the use of radiolabeled compounds or time-intensive tandem quadrupole analysis. Methods were simultaneously developed for a wide variety of compounds from different chemical classes. Though many compounds would not fit the generic method and would require additional set-up time, sufficient time is saved to make this workflow suitable for substrate and/or inhibition assays early in the drug discovery process.