Investigation of Fluorescein Derivatives as Substrates of Organic Anion Transporting Polypeptide (OATP) 1B1 To Develop Sensitive Fluorescence-Based OATP1B1 Inhibition Assays

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• 作者：Saki Izumi ; Yoshitane Nozaki ; Takafumi Komori ; Osamu Takenaka ; Kazuya Maeda ; Hiroyuki Kusuhara ; Yuichi Sugiyama
• 刊名：Molecular Pharmaceutics
• 出版年：2016
• 出版时间：February 1, 2016
• 年：2016
• 卷：13
• 期：2
• 页码：438-448
• 全文大小：663K
• ISSN：1543-8392

文摘

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of various drugs. Because OATP1B1 is a site of drug–drug interactions (DDIs), evaluating the inhibitory potential of drug candidates on OATP1B1 is required during drug development. For establishing a highly sensitive, high-throughput fluorescence-based OATP1B1 inhibition assay system, the present study focused on fluorescein (FL) and its derivatives and evaluated their uptake via OATP1B1 as well as OATP1B3 and OATP2B1 using the transporter-expressing human embryonic kidney 293 cells. We identified 2′,7′-dichlorofluorescein (DCF), 4′,5′-dibromofluorescein (DBF), and Oregon green (OG) as good OATP1B1 substrates with K_m values of 5.29, 4.16, and 54.1 μM and V_max values of 87.9, 48.1, and 187 pmol/min/mg protein, respectively. In addition to FL, fluo-3, and 8-fluorescein-cAMP, OG, and DBF were identified as OATP1B3 substrates. FL, OG, DCF, and DBF were identified as OATP2B1 substrates. Among the FL derivatives, DCF displayed the highest OATP1B1-mediated uptake. The K_i values of 14 compounds on OATP1B1 determined with DCF as a probe exhibited good agreement with those obtained using [³H]estradiol-17β-glucuronide (E₂G) as a substrate, whereas [³H]estrone-3-sulfate and [³H]sulfobromophthalein yielded higher K_i values for all inhibitors than DCF. Mutually competitive inhibition observed between DCF and E₂G suggested that they share the same binding site on OATP1B1. Therefore, DCF as well as E₂G can be used as sensitive probes for in vitro OATP1B1 inhibition assays, which will help mitigate the risk of false-negative DDI predictions potentially caused by substrate-dependent K_i variations.