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Pregnancy results in significant changes in drug pharmacokinetics (PK). While previous studies have elucidated the impact of pregnancy-related hormones (PRH) on mRNA or protein expression and activity of major hepatic metabolizing enzymes, their effect on hepatic drug transporters remains largely unexplored. Therefore, we investigated the effect of a cocktail of PRH on the mRNA expression and activity of hepatic transporters. Plated human hepatocytes (PHH) from 3 premenopausal donors were incubated, in triplicate, for 72 h, with vehicle (DMSO < 0.01%), rifampin (10 μM; positive control) or a cocktail of PRH consisting of estrone, estradiol, estriol, estetrol, progesterone, cortisol, testosterone, oxytocin, and placental growth hormone. The PRH concentrations replicated 0.1×, 1×, or 10× of the plasma concentrations of these hormones observed during each of the three trimesters of pregnancy. After treatment, mRNA expression (quantified by qPCR) of hepatic influx and efflux transporters as well as the activity of influx transporters was quantified (uptake of a selective substrate ± corresponding transporter inhibitor). The data were expressed relative to that in the control (vehicle) group. Significance was evaluated by ANOVA (followed by Dunn's multiple comparisons) or unpaired -test when the within-lot data were analyzed, or repeated measures ANOVA (followed by Dunn's multiple comparisons) or paired -test when data from all 3 lots were analyzed ( < 0.05). In general, a) PRH cocktails significantly induced transporter mRNA expression in the following order OAT2 ≈ NTCP ≈ OCT1 > OATP2B1 and repressed mRNA expression in the following order OATP1B3 > OATP1B1; b) these changes translated into significant induction of OAT2 (T1-T3) and NTCP (T2-T3, in only two lots) activity at the 1× PRH concentration. Compared with the influx transporters, the induction of mRNA expression of efflux transporters was modest, with mRNA expression of MRP2 and BSEP being induced the most. Once these data are verified through probe drug PK studies in pregnancy, they can be populated into physiologically based pharmacokinetic (PBPK) models to predict, for all trimesters of pregnancy, transporter-mediated clearance of any drug that is a substrate of the affected transporters.

Chemotherapy resistance is a significant barrier to effective cancer treatment. A key mechanism of resistance at the single-cell level is the overexpression of drug transporters in the ABC family, particularly P-glycoprotein (P-gp), which leads to multidrug resistance (MDR). Inhibitors of these transporters can help re-sensitize cancer cells to chemotherapeutics. This study evaluated elacridar (GG918 and GF120918), a potent third-generation P-gp inhibitor, for its ability to reverse MDR in paclitaxel (PAC)-resistant ovarian cancer cell lines. Sensitive and PAC-resistant cells were cultured in two-dimensional (2D) and three-dimensional (3D) models. MDR1 gene expression was analyzed using Q-PCR, and P-gp protein expression was examined via Western blot and immunofluorescence. Drug sensitivity was evaluated with MTT assays, and P-gp activity was analyzed by flow cytometry and fluorescence microscopy. Elacridar effectively inhibited P-gp activity and increased sensitivity to PAC and doxorubicin (DOX) in 2D cultures but not cisplatin (CIS). In 3D spheroids, P-gp activity inhibition was observed via Calcein-AM staining. However, no re-sensitization to PAC occurred and limited improvement was observed for DOX. These findings suggest that elacridar effectively inhibits P-gp in both 2D and 3D conditions. However, its ability to overcome drug resistance in 3D models is limited, highlighting the complexity of tissue-specific resistance mechanisms.

Chemotherapy resistance is a major obstacle in the treatment of ovarian cancer, often resulting in disease recurrence and poor prognosis for patients. A key contributor to this resistance is the overexpression of ATP-binding cassette (ABC) transporters, including breast cancer resistance protein (BCRP/ABCG2), which actively effluxes chemotherapeutic agents such as topotecan (TOP) or mitoxantrone (MIT), limiting their intracellular accumulation and efficacy. This study investigated the potential of elacridar (GG918), a potent dual P-gp and BCRP inhibitor, to overcome drug resistance in ovarian cancer cell lines. Both TOP-sensitive and TOP-resistant ovarian cancer cells were grown in two-dimensional (2D) monolayers and three-dimensional (3D) spheroid models to better mimic the tumor microenvironment. The expression of the ABCG2 gene was quantified via qPCR and BCRP protein levels were assessed by western blotting and immunofluorescence. Drug response was evaluated using MTT viability assays, while BCRP transporter activity was examined using flow cytometry and microscopic assessment of the intracellular retention of BCRP fluorescent substrates (Hoechst 33342 and MIT). In both 2D and 3D cultures, elacridar effectively inhibited BCRP function and significantly enhanced sensitivity to TOP. These findings suggest that elacridar can inhibit BCRP-mediated drug resistance in ovarian cancer cell models.

Objective The influence of obesity on renal drug clearance (CLr) remains difficult to predict. This study quantifies obesity-related alterations in CLr for drugs eliminated via glomerular filtration (GF/CL GF ) and active tubular secretion (ATS/CL ATS ) and assesses the systematic accuracy of dosing based on allometric scaling with an exponent of 0.75 or flat dosing (exponent of 0). Methods A physiologically-based pharmacokinetic (PBPK) approach was used to simulate CL GF and CL ATS for 11,520 hypothetical drugs in typical subjects with body mass index (BMI) between 20 and 60. Correlations between changes in CL GF and CL ATS and subject or drug properties were investigated. Moreover, for each drug, CLr values scaled to individuals with obesity from CLr values in normal-weight individuals were compared to PBPK predictions of CLr. Systematic scaling accuracy was defined as the prediction error being less than ± 30% for all drugs. Results CLr through GF and ATS increased with BMI, albeit to different extents, depending on drug properties. When BMI was below 30 kg/m 2 and transporter activity remained unchanged, the CLr between subjects of normal weight and with overweight or obesity differed less than 30% and both scaling methods were systematically accurate. For individuals with higher BMI, drug properties need to be taken into account when defining scenarios of systematic scaling accuracy. Conclusion In individuals with a BMI above 30 kg/m 2 , neither 0.75 allometric scaling nor no scaling (flat dosing) is systematically accurate for renally cleared drugs. Strategies are provided to define systematic scaling accuracy a priori , based on subject and drug properties.

Excitatory amino acid transporter 2 (EAAT2), the gene of which is known as solute carrier family 1 member 2 (SLC1A2), is an important membrane‐bound transporter that mediates approximately 90% of the transport and clearance of l‐glutamate at synapses in the central nervous system (CNS). Transmembrane domain 2 (TM2) of EAAT2 is close to hairpin loop 2 (HP2) and far away from HP1 in the inward‐facing conformation. In the present study, 14 crucial amino acid residues of TM2 were identified via alanine‐scanning mutations. Further analysis in EAAT2‐transfected HeLa cells in vitro showed that alanine substitutions of these residues resulted in a decrease in the efficiency of trafficking/targeting to the plasma membrane and/or reduced functionality of membrane‐bound, which resulted in impaired transporter activity. After additional mutations, the transporter activities of some alanine‐substitution mutants recovered. Specifically, the P95A mutant decreased EAAT2‐associated anion currents. The Michaelis constant (Km) values of the mutant proteins L85A, L92A and L101A were increased significantly, whereas R87 and P95A were decreased significantly, indicating that the mutations L85A, L92A and L101A reduced the affinity of the transporter and the substrate, whereas R87A and P95A enhanced this affinity. The maximum velocity (Vmax) values of all 14 alanine mutant proteins were decreased significantly, indicating that all these mutations reduced the substrate transport rate. These results suggest that critical residues in TM2 affect not only the protein expression and membrane‐bound localization of EAAT2, but also its interactions with substrates. Additionally, our findings elucidate that the P95A mutant decreased EAAT2‐related anion currents. Our results indicate that the TM2 of EAAT2 plays a vital role in the transport process. The key residues in TM2 affect protein expression in the membrane, substrate transport and the anion currents of EAAT2.

Phytohormones are a group of small chemical molecules that play vital roles in plant development, metabolism, and stress responses. Phytohormones often have distinct biosynthesis and signaling perception sites, requiring long- or short-distance transportation. Unlike biosynthesis and signal transduction, phytohormone transport across cells and organs is poorly understood. The transporter activity assay is a bottleneck for the functional characterization of novel phytohormone transporters. In the present study, we report a tobacco syringe agroinfiltration and liquid chromatography tandem mass spectrometry (TSAL)-based method for performing a phytohormone transporter activity assay using endogenous hormones present in tobacco ( Nicotiana benthamiana ) leaves. A transporter activity assay using this method does not require isotope-labeled substrates and can be conveniently performed for screening multiple substrates by using endogenous hormones in tobacco leaves. The transporter activities of three known hormone transporters, namely AtABCG25 for abscisic acid, AtABCG16 for jasmonic acid, and AtPUP14 for cytokinin, were all successfully validated using this method. Using this method, cytokinins were found to be the preferred substrates of an unknown maize ( Zea mays ) transporter ZmABCG43. ZmABCG43 transporter activities toward cytokinins were confirmed in a cytokinin long-distance transport mutant atabcg14 through gene complementation. Thus, the TSAL method has the potential to be used for basic substrate characterization of novel phytohormone transporters or for the screening of novel transporters for a specific phytohormone on a large scale.

The functional characterization of plasma membrane transport proteins often relies on their heterologous expression in cultured cells. However, some transporters exhibit low activity, hindering meaningful functional assays. Heterologous expression is usually based on strong viral promoters which in living cells are prone to promoter silencing, a major problem. Here, we investigated the efficacy of low‐cost histone deacetylase (HDAC) inhibitors in enhancing transporter activity, comparing the established sodium butyrate (the sodium salt of butyric acid) with valproate/valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA, also known as vorinostat). Using 293 cells stably transfected with pEBTet plasmids containing the CMV promotor to express the transporters SLC16A9, SLC22A15, and OATP1A2, we measured substrate efflux or uptake via LC–MS/MS following overnight preincubation with the HDAC inhibitors. All three compounds markedly stimulated transporter activity. VPA was less effective than butyrate but still surpassed control conditions. SAHA was cytotoxic at 6 μm, but at 2 μm, the enhancement was consistently comparable to 5 mm butyrate. Additionally, SAHA was more cost‐effective and devoid of the repulsive odor characteristic of butyrate. Our findings advocate for replacing butyrate with SAHA to enhance heterologously expressed transporter activity. This offers a more efficient and user‐friendly alternative for functional assays. Heterologous expression of membrane transporters in cultured cells is essential for functional characterization, but is sometimes limited by low activity. Our study compares the HDAC inhibitors butyrate, VPA and SAHA to enhance transport activity. We propose to replace butyrate by SAHA: it is equally effective, devoid of repulsive odor, costs less, and the required concentration is 2500 times lower.

Fat accumulation (steatosis) in ballooned hepatocytes alters the expression of membrane transporters in Zucker fatty (fa/fa) rats. The aim of the study was to quantify the functions of these transporters and their impact on hepatocyte concentrations using a clinical hepatobiliary contrast agent (Gadobenate dimeglumine, BOPTA) for liver imaging. In isolated and perfused rat livers, we quantified BOPTA accumulation and decay profiles in fa/+ (normal) and fa/fa hepatocytes by placing a gamma counter over livers. Profiles of BOPTA accumulation and decay in hepatocytes were analysed with nonlinear regressions to characterise BOPTA influx and efflux across hepatocyte transporters. At the end of the accumulation period, BOPTA hepatocyte concentrations and influx clearances were not significantly different in fa/+ and fa/fa livers. In contrast, bile clearance was significantly lower in fatty hepatocytes while efflux clearance back to sinusoids compensated the low efflux into canaliculi. The time when BOPTA cellular efflux impacts the accumulation profile of hepatocyte concentrations was slightly delayed (2 min) by steatosis, anticipating a delayed emptying of hepatocytes. The experimental model is useful for quantifying the functions of hepatocyte transporters in liver diseases.

Hyperuricemia, a lifestyle-related disease characterized by elevated serum urate levels, is the main risk factor for gout; therefore, the serum urate-lowering effects of human diets or dietary ingredients have attracted widespread interest. As Urate transporter 1 (URAT1) governs most urate reabsorption from primary urine into blood, URAT1 inhibition helps decrease serum urate levels by increasing the net renal urate excretion. In this study, we used a cell-based urate transport assay to investigate the URAT1-inhibitory effects of 162 extracts of plant materials consumed by humans. Among these, we focused on Aspalathus linearis, the source of rooibos tea, to explore its active ingredients. Using liquid–liquid extraction with subsequent column chromatography, as well as spectrometric analyses for chemical characterization, we identified quercetin as a URAT1 inhibitor. We also investigated the URAT1-inhibitory activities of 23 dietary ingredients including nine flavanols, two flavanonols, two flavones, two isoflavonoids, eight chalcones, and a coumarin. Among the tested authentic chemicals, fisetin and quercetin showed the strongest and second-strongest URAT1-inhibitory activities, with IC50 values of 7.5 and 12.6 μM, respectively. Although these effects of phytochemicals should be investigated further in human studies, our findings may provide new clues for using nutraceuticals to promote health.