Department of Pharmaceutics, School of Pharmacy, Aichi Gakuin University, 1-100 Kusumoto, Chikusa-ku, Nagoya, 464-8650, Japan.
Pharm Res. 2020 Jun 1;37(6):115. doi: 10.1007/s11095-020-02831-x.
In this study, we investigated organic anion transporting polypeptide 2B1 (OATP2B1)-mediated uptake of fluorescent anions to better identify fluorescent substrates for in vitro OATP2B1 assays. The OATP2B1 is involved in the intestinal absorption and one of the pharmacokinetic determinants of orally administered drugs.
A microplate reader was used to determine the cellular accumulation of the fluorescent compounds into the OATP2B1 or the empty vector-transfected HEK293 cells.
Two types of derivatives were found to be OATP2B1 substrates: heavy halogenated derivatives, such as 4',5'-dibromofluorescein (DBF), and carboxylated derivatives, such as 5-carboxyfluorescein (5-CF). The DBF and 5-CF were transported in a time and concentration-dependent manner. The DBF was transported at a broad pH (pH 6.5-8.0) while 5-CF was transported at an acidic pH (pH 5.5-6.5). The K values were 0.818 ± 0.067 μM at pH 7.4 for DBF and 8.56 ± 0.41 μM at pH 5.5 for 5-CF. The OATP2B1 inhibitors, including atorvastatin, bromosulfophthalein, glibenclamide, sulfasalazine, talinolol, and estrone 3-sulfate, inhibited the DBF and the 5-CF transport. Contrastively, testosterone, dehydroepiandrosterone sulfate, and progesterone inhibited the DBF transport but stimulated the 5-CF transport. Natural flavonoid aglycones, such as naringenin and baicalein, also exhibited substrate-dependent effects in this manner.
We found two fluorescein analogs, DBF and 5-CF as the OATP2B1 substrates that exhibited substrate-dependent interactions.
在这项研究中，我们研究了有机阴离子转运多肽 2B1（OATP2B1）介导的荧光阴离子摄取，以更好地鉴定用于体外 OATP2B1 测定的荧光底物。OATP2B1 参与肠道吸收，是口服药物的药代动力学决定因素之一。
使用微孔板读数器确定荧光化合物进入 OATP2B1 或空载体转染的 HEK293 细胞的细胞内积累。
发现两种类型的衍生物是 OATP2B1 的底物：重卤素衍生物，如 4',5'-二溴荧光素（DBF），和羧基衍生物，如 5-羧基荧光素（5-CF）。DBF 和 5-CF 以时间和浓度依赖的方式转运。DBF 在较宽的 pH 值（pH 6.5-8.0）下转运，而 5-CF 在酸性 pH 值（pH 5.5-6.5）下转运。在 pH 7.4 时，DBF 的 K 值为 0.818 ± 0.067 μM，在 pH 5.5 时，5-CF 的 K 值为 8.56 ± 0.41 μM。OATP2B1 抑制剂，包括阿托伐他汀、溴磺酞、格列本脲、柳氮磺胺吡啶、他林洛尔和雌酮 3-硫酸盐，抑制 DBF 和 5-CF 的转运。相比之下，睾丸酮、脱氢表雄酮硫酸盐和孕酮抑制 DBF 转运但刺激 5-CF 转运。天然类黄酮苷元，如柚皮苷和黄芩素，也以这种方式表现出底物依赖性的作用。
我们发现两种荧光素类似物，DBF 和 5-CF 作为 OATP2B1 的底物，表现出底物依赖性相互作用。
