Development of a fluorescence-based assay for screening of modulators of human Organic Anion Transporter 1B3 (OATP1B3)

Baldes, Christiane; Koenig, Petra; Neumann, Dirk; Lenhof, Hans Peter; Kohlbacher, Oliver; Lehr, Claus‐Michael

doi:10.1016/j.ejpb.2005.06.001

Cited by 39 publications

(25 citation statements)

References 13 publications

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“…These include: 22 and 9.3 :M for CDF 15 and CGamF, 21 respectively, in sandwich-cultured rat hepatocytes; 4.6 :M for CLF 22 in OATP1B3-transfected CHO cells; 2.3, 3.1, and 6.8 :M for Fluo-3 in OATP1B1-and OATP1B3-transfected CHO cells 44 and in HEK-OATP1B3 cells. 16 Uptake rates of NaFluo and other known OATP substrates (ES, EG, and CGamF) in OATP1B1/3-transfected cells were compared by measuring uptake at a substrate concentration of 1 :M (<K m values of substrates tested). 34,44 Figure 6 illustrates that under these nonsaturating conditions, NaFluo behaves as a nonspecific OATP1B substrate.…”

Section: Discussionmentioning

confidence: 99%

“…15 Fluo-3, a fluorescent calcium indicator, 8-fluorescein-cyclic adenosine monophosphate, and fluorescein-methotrexate have been used in fluorescence-based assays for screening of OATP modulators. 16,17,18 Furthermore, several fluorescent bile salt derivatives have been synthesized. Chenodeoxycholic acid was linked with fluorescent 7-nitrobenz-2-oxa-1,3-diazole and the uptake of this conjugate was characterized in HepG2 cells.…”

Section: Introductionmentioning

confidence: 99%

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Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

Bruyn

Fattah

Stieger

et al. 2011

Journal of Pharmaceutical Sciences

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

Bruyn

Fattah

Stieger

et al. 2011

Journal of Pharmaceutical Sciences

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“…In vitro methods for assessing OATP1B-mediated hepatic drug uptake based on primary human hepatocytes are generally predictive of the in vivo situation but remain resourceintensive. Hence, several studies have successfully relied on the use of higher throughput assays with transfected human embryonic kidney (HEK)293 or Chinese hamster ovary (CHO) cells to investigate OATP1B inhibition and transporter kinetics (Baldes et al, 2006;Annaert et al, 2010;Bednarczyk, 2010;Gui et al, 2010). These studies led to the identification of new OATP1B inhibitors and/or the characterization of the inhibition potential of existing drugs (e.g., HIV protease inhibitors) (Annaert et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

Structure-Based Identification of OATP1B1/3 Inhibitors

Bruyn

IJzerman

Stieger

et al. 2013

Molecular Pharmacology

119

109

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Several recent studies show that inhibition of the hepatic transport proteins organic anion-transporting polypeptide 1B1 (OATP1B1) and 1B3 (OATP1B3) can result in clinically relevant drug-drug interactions (DDI). To avoid late-stage development drug failures due to OATP1B-mediated DDI, predictive in vitro and in silico methods should be implemented at an early stage of the drug candidate evaluation process. In the present study, we first developed a high-throughput in vitro transporter inhibition assay for the OATP1B subfamily. A total of 2000 compounds were tested as potential modulators of the uptake of the OATP1B substrate sodium fluorescein, in OATP1B1-or 1B3-transfected Chinese hamster ovary cells. At an equimolar substrate-inhibitor concentration of 10 mM, 212 and 139 molecules were identified as OATP1B1 and OATP1B3 inhibitors, respectively (minimum 50% inhibition). For 69 compounds, previously not identified as OATP1B inhibitors, concentrationdependent inhibition was also determined, yielding K i values ranging from 0.06 to 6.5 mM. Based on these in vitro data, we subsequently developed a proteochemometrics-based in silico model, which predicted OATP1B inhibitors in the test group (20% of the dataset) with high specificity (86%) and sensitivity (78%). Moreover, several physicochemical compound properties and substructures related to OATP1B1/1B3 inhibition or inactivity were identified. Finally, model performance was prospectively verified with a set of 54 compounds not included in the original dataset. This validation indicated that 80 and 74% of the compounds were correctly classified for OATP1B1 and OATP1B3 inhibition, respectively.

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“…In addition to this important role in normophysiology, atopic expression of drug transport proteins is observed in many tumours [16], [17]; for example, increased expression of MDR1 is associated with a reduced response to therapy in breast cancer [18], presumably due to increased efflux of chemotherapeutic agents from the tumour cells. Given the need to better understand, and quantify, transport processes, tracer dyes are being increasingly used to examine these processes, with R123, Fluo-3 and carboxydichlorofluroscein being increasingly used to study MDR1-, OATP1B3- and MRP2- mediated transport, respectively [4], [19], [20]. Herein, we examine the physiochemical properties of R123, and characterize the optimal conditions for its use as a tracer dye to quantify MDR1-mediated transport.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

et al. 2012

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Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.

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Development of a fluorescence-based assay for screening of modulators of human Organic Anion Transporter 1B3 (OATP1B3)

Cited by 39 publications

References 13 publications

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

Structure-Based Identification of OATP1B1/3 Inhibitors

Characterization of Rhodamine-123 as a Tracer Dye for Use In In vitro Drug Transport Assays

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