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Characterization of a Novel “Thaw and Go” Cell‐based Transporter Model for Studying SLC Transporters
Author(s) -
Wang Jie,
Bourgea Joanne,
Cooper Kirsten,
Patten Christopher,
Li Na
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.lb527
Subject(s) - transporter , organic anion transporter 1 , chemistry , estrone sulfate , hek 293 cells , pharmacology , substrate (aquarium) , incubation , biochemistry , biology , estrone , gene , ecology , hormone
Objective Development and characterization of a novel cell‐based transporter model to study regulatory agencies (USFDA/EMA) recommended and industry important SLC transporters. Methods Cryopreserved HEK293 cells which transiently overexpress OATP1B1*1a (wild‐type), OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2‐K, OATP1A2, OATP2B1, PEPT1, PEPT2, NTCP and empty vector, respectively, were thawed and plated on 24‐well Poly‐D‐Lysine coated plates, and then refed with plating media in the presence or absence of 2mM sodium butyrate at 3‐4 hours post plating. Uptake assays were performed at 24 hrs post plating. Following two washes, cells were pre‐equilibrated with HBSS buffer for 10min, MATE1 and MATE2‐K cells require extra incubation with HBSS buffer containing NH 4 Cl. The cells were then incubated with substrate or inhibitor for desired amount of time. After the assay was terminated, the cells were lysed and subjected to analysis. Results The novel SLC transporter model, including OATP1B1*1a (wild‐type), OATP1B3, OAT1, OAT3, OCT1, OCT2, MATE1, MATE2‐K, OATP1A2, OATP2B1, PEPT1, PEPT2, and NTCP cells, was characterized for time and substrate concentration‐dependent uptake (Km and Vmax determinations) using prototypical substrates as well as known drug substrates. The model was also validated using selected prototypical transporter inhibitors recommended by regulatory agencies. The kinetic profiles of the selected substrates and IC 50 of selected inhibitors were comparable to the published literature and also consistent across independent laboratories. As an example, OATP1B1*1a Km comparison using various substrates is demonstrated below.OATP1B1*1a Substrates Estradiol‐17β‐Glucuronide Estrone‐3‐Sulfate Rosuvastatin Fluorescein MethotrexateK m (µM) using Corning® OATP1B1*1a Cells 6.2 0.4 14.2 5.2K m (µM) reported in literature 6.3 0.46 13.1 3.8Conclusions The data demonstrated this new SLC transporter model is a validated and compliant in vitro tool to study drug interactions with SLC transporters recommended by regulatory agencies (USFDA/EMA) and industry. This novel cell‐based transporter model eliminates the need for intensive cell culture and maintenance, as well as providing consistent lot‐to‐lot performance.