Background and Objectives Citrus flavonoids are not only components of daily nutrition, they are also promoted as dietary supplements and are important ingredients in traditional medicines. Interactions of flavonoids with synthetic drugs represent an often neglected issue. We therefore investigated in vitro whether the polymethoxyflavones nobiletin, sinensetin, and tangeretin and the flavonoid rutinosides didymin, hesperidin, and narirutin can inhibit human organic anion transporting polypeptides (OATP) 1B1, 1B3, and 2B1, which are important transporters mediating drug-drug and food-drug interactions. Methods Inhibition was investigated by quantifying the decreased uptake of the fluorescent OATP1B1 and OATP1B3 substrate 8-fluorescein-cAMP in HEK293 cells overexpressing OATP1B1 or OATP1B3 and of the fluorescent OATP2B1 substrate 4′,5′-dibromofluorescein in HEK293 cells overexpressing OATP2B1. Results We demonstrate that all flavonoids investigated inhibit OATP2B1 in the lower micromolar range (IC₅₀ between 1.6 and 14.2 µM), but only the polymethoxyflavones also inhibit OATP1B1 and 1B3 (IC₅₀ between 2.1 and 21 µM). Conclusions All flavonoids investigated might contribute to the intestinal OATP2B1-based interactions with drugs observed with citrus juices or fruits. In contrast, the concentration of the polymethoxyflavones after consumption of citrus juices or fruits is most likely too low to reach relevant systemic concentrations and thus to inhibit hepatic OATP1B1 and OATP1B3, but there might be a risk when they are consumed as medicines or as dietary supplements.

(1) Background: Hydroxychloroquine is used to treat malaria and autoimmune diseases, and its potential use against COVID-19 is currently under investigation. Thus far, information on interactions of hydroxychloroquine with drug transporters mediating drug-drug interactions is limited. We assessed the inhibition of important efflux (P-glycoprotein (P-gp), breast cancer resistance protein (BCRP)) and uptake transporters (organic anion transporting polypeptide (OATP)-1B1, OATP1B3, OATP2B1) by hydroxychloroquine, tested its P-gp and BCRP substrate characteristics, and evaluated the induction of pharmacokinetically relevant genes regulated by the nuclear pregnane X (PXR) (CYP3A4, ABCB1) and aryl hydrocarbon receptor (AhR) (CYP1A1, CYP1A2). (2) Methods: Transporter inhibition was evaluated in transporter over-expressing cell lines using fluorescent probe substrates. P-gp and BCRP substrate characteristics were assessed by comparing growth inhibition of over-expressing and parental cell lines. Possible mRNA induction was analysed in LS180 cells by quantitative real-time PCR. (3) Results: Hydroxychloroquine did not inhibit BCRP or the OATPs tested but inhibited P-gp at concentrations exceeding 10 µM. P-gp overexpressing cells were 5.2-fold more resistant to hydroxychloroquine than control cells stressing its substrate characteristics. Hydroxychloroquine did not induce genes regulated by PXR or AhR. (4) Conclusions: This is the first evidence that hydroxychloroquine’s interaction potential with drug transporters is low, albeit bioavailability of simultaneously orally administered P-gp substrates might be increased by hydroxychloroquine.

The solute carrier L-type amino acid transporter 1 (LAT-1/SLC7A5) is a viable target for drug delivery to the central nervous system (CNS) and tumors due to its high abundance at the blood–brain barrier and in tumor tissue. LAT-1 is only localized on the cell surface as a heterodimer with CD98, which is not required for transporter function. To support future CNS drug-delivery development based on LAT-1 targeting, we established an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) assay for stable isotopically labeled leucine ([¹³C₆, ¹⁵N]-L-leucine), with a dynamic range of 0.1–1000 ng/mL that can be applied for the functional testing of LAT-1 activity when combined with specific inhibitors and, consequently, the LAT-1 inhibition capacity of new compounds. The assay was established in a 96-well format, facilitating high-throughput experiments, and, hence, can support the screening for novel inhibitors. Applicable recommendations of the US Food and Drug Administration and European Medicines Agency for bioanalytical method validation were followed to validate the assay. The assay was applied to investigate the IC₅₀ of two well-known LAT-1 inhibitors on hCMEC/D3 cells: the highly specific LAT-1 inhibitor JPH203, which was also used to demonstrate LAT-1 specific uptake, and the general system L inhibitor BCH. In addition, the [¹³C₆, ¹⁵N]-L-leucine uptake was determined on two human brain capillary endothelial cell lines (NKIM-6 and hCMEC/D3), which were characterized for their expressional differences of LAT-1 at the protein and mRNA level and the surface amount of CD98. The IC₅₀ values of the inhibitors were in concordance with previously reported values. Furthermore, the [¹³C₆, ¹⁵N]-L-leucine uptake was significantly higher in hCMEC/D3 cells compared to NKIM-6 cells, which correlated with higher expression of LAT-1 and a higher surface amount of CD98. Therefore, the UPLC-MS/MS quantification of ([¹³C₆, ¹⁵N]-L-leucine is a feasible strategy for the functional characterization of LAT-1 activity in cells or tissue.

The human peptide transporter hPepT1 (SLC15A1), physiologically transporting dipeptides and tripeptides generated during food digestion, also plays a role in the uptake of small bioactive peptides and peptide-like drugs. Moreover, it might be addressed in prodrug strategies of poorly absorbed drugs. We hypothesised that the cyclic drug peptides octreotide and pasireotide could be substrates of this transporter because their diameter can resemble the size of dipeptides or tripeptides due to their strong structural curvature and because they reach the systemic circulation in Beagle dogs. For investigating possible hPepT1 substrate characteristics, we generated and characterised a CHO-K1 cell line overexpressing SLC15A1 by transfection and selection via magnetic beads. Possible inhibition of the uptake of the prototypical substrate Gly-Sar by octreotide and pasireotide was screened, followed by quantifying the uptake of the cyclic peptides in cells overexpressing SLC15A1 compared with the parental cell line. Although inhibition of Gly-Sar uptake was observed, uptake of octreotide and pasireotide was not increased in SLC15A1 overexpressing cells, indicating a lack of transport by hPepT1. Our data clearly indicate that octreotide and pasireotide are nonsubstrate inhibitors of hPepT1 and that their oral bioavailability cannot be explained by absorption via hPepT1.