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

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 <i>K</i>_m values of 5.29, 4.16, and 54.1 μM and <i>V</i>_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 <i>K</i>_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 <i>K</i>_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 <i>K</i>_i variations

Relative Activity Factor (RAF)-Based Scaling of Uptake Clearance Mediated by Organic Anion Transporting Polypeptide (OATP) 1B1 and OATP1B3 in Human Hepatocytes

In vitro–in vivo extrapolation based on uptake clearance determined in human hepatocytes has been used to predict in vivo hepatic clearance of organic anion transporting polypeptide (OATP) substrates. This study evaluated the relative activity factor (RAF) approach to extrapolate active uptake clearance in transporter-transfected cell systems (CL_uptake) to that in human hepatocyte suspensions (PS_inf,act). RAF values for OATP1B1 and OATP1B3 were determined in two batches of cryopreserved human hepatocytes using estrone-3-sulfate and cholecystokinin octapeptide as reference substrates, respectively. Fourteen OATP1B substrate drugs selected (atorvastatin, bosentan, cerivastatin, fexofenadine, fluvastatin, glibenclamide, irbesartan, nateglinide, pitavastatin, pravastatin, rosuvastatin, telmisartan, torasemide, and valsartan) showed temperature-dependent uptake in human hepatocytes. In transporter-transfected cells, OATP1B1- and OATP1B3-mediated uptake was observed in all compounds except for telmisartan. RAF-based net CL_uptake was mainly accounted for by OATP1B1 (72.3–99.7%) and fell within the 3-fold of PS_inf,act observed in human hepatocytes in 11 out of 13 compounds (excluding telmisartan). This study demonstrated that the RAF approach provides a quantitative index of OATP1B1- and OATP1B3-mediated PS_inf,act in human hepatocytes, which will facilitate the optimization of the pharmacokinetic properties of OATP1B substrates at nonclinical stages of drug development

Effect of Knockout of <i>Mdr1a</i> and <i>Mdr1b</i> ABCB1 Genes on the Systemic Exposure of a Doxorubicin-Conjugated Block Copolymer in Mice

We previously elucidated that ATP-binding cassette subfamily B member 1 (ABCB1) mediates the efflux of doxorubicin-conjugated block copolymers from HeLa cells. Here, we investigated the role of ABCB1 in the in vivo behavior of a doxorubicin-conjugated polymer in <i>Mdr1<i>a</i>/1b­(−/−)</i> mice. The area under the curve for intravenously administered polymer in <i>Mdr1<i>a</i>/1b­(−/−)</i> mice was 2.2-fold greater than that in wild-type mice. The polymer was mostly distributed in the liver followed by spleen and less so in the brain, heart, kidney, and lung. The amount of polymer excreted in the urine was significantly decreased in <i>Mdr1<i>a</i>/1b­(−/−)</i> mice. The amounts of polymers excreted in the feces were similar in both groups despite the higher systemic exposure in <i>Mdr1<i>a</i>/1b­(−/−)</i> mice. Confocal microscopy images showed polymer localized in CD68⁺ macrophages in the liver. These results show that knockout of ABCB1 prolonged systemic exposure of the doxorubicin-conjugated polymer in mice. Our results suggest that ABCB1 mediated the excretion of doxorubicin-conjugated polymer in urine and feces. Our results provide valuable information about the behavior of block copolymers in vivo, which is important for evaluating the pharmacokinetics of active substances conjugated to block copolymers or the accumulation of block copolymers in vivo