Ting Chan

The inhibitory effects of the bioactive components isolated from Scutellaria baicalensis on the cellular uptake mediated by the essential solute carrier transporters.

Solute carrier transporters (SLCs), in particular the organic anion transporters (OATs), OAT polypeptides (OATPs), and organic cation transporters (OCTs/OCTNs), are the important membrane proteins responsible for the cellular influx of various drugs. Baicalein (BA), baicalin (BG), and wogonin (WG) are the three major bioactive components of Scutellaria baicalensis. In this study, we evaluated the inhibitory effects of BA, BG, and WG on the cellular uptake of specific substrates mediated by the essential SLCs in human embryonic kidney-293 cells. Our data demonstrated that BA and WG significantly inhibit the OAT1-, OAT3-, and OATP1B3-mediated uptake; BG effectively reduces the influx of substrates of OAT3, OAT4, OATP1B3, and OATP2B1; WG is a potent inhibitor of OCT3. Our further kinetic analysis derived the IC50 values of these compounds with pronounced inhibitory effects on SLCs, particularly the inhibitions of WG on OAT1 and OCT3 and that of BA and WG on OAT3. Our study comprehensively evaluated the inhibitory effects of three bioactive components of Scutellaria baicalensis on the uptake of specific substrates mediated by the essential SLC transporters, which suggested that precautions will be needed when coadministrating drugs with Scutellaria baicalensis so as to prevent the unfavorable drug-drug/herb interactions in human.

Selective inhibition of human solute carrier transporters by multikinase inhibitors

Solute carrier (SLC) transporters regulate the cellular influx and disposition of endogenous and xenobiotic compounds, including anticancer agents such as the multikinase inhibitors (MKIs). Recent evidence suggests that MKIs may also inhibit SLC-dependent transport of coadministered drugs, although present information on the relative susceptibilities of multiple SLC transporters is limited. This study evaluated 18 MKI drugs and metabolites as inhibitors of prototypic substrate uptake by 13 SLC transporters that were overexpressed in human embryonic kidney cells. Organic anion transporting polypeptides (OATPs) 1A2, 1B3, and 2B1, organic anion transporter 3 (OAT3), and organic cation transporter 1 (OCT1) were inhibited by most MKIs, whereas substrate uptake by OATP1B1, OAT1, 2, and 4, OCT2 and 3, and organic zwitterion/cation transporter 1 (OCTN1) was less susceptible to inhibition; OCTN2 was also inhibited by cediranib. In further studies, IC50 values were determined for the most effective MKIs, and erlotinib and cediranib were found to be potent competitive inhibitors of OATP2B1 (Ki = 41 nM) and OATP1A2 (Ki = 33 nM), respectively. From predictive approaches, several MKI-SLC interactions were found to be of potential in vivo significance.

Interaction of the Bioactive Flavonol, Icariin, with the Essential Human Solute Carrier Transporters

Solute carrier transporters (SLCs), in particular the organic anion transporting polypeptides (OATPs) and organic anion/cation transporters (OATs/OCTs), are responsible for the cellular entry of many clinically important drugs in body. They largely influence drug safety and efficacy. Icariin is a flavonol widely present in many herbal preparations, which is used to improve sexual function and prevent osteogenesis. However, precautions are necessary in therapies containing icariin due to its involvement in drug–drug/herb interactions, possibly mediated through competing drug uptake via membrane‐transporter proteins. This study is the first to comprehensively evaluate the interactions between icariin and a range of essential SLCs. Our data demonstrated that icariin can significantly inhibit OATP1B3‐ and OATP2B1‐mediated cellular uptake of specific substrates (IC50 of 3.0 ± 1.3 and 6.4 ± 1.9 μM, respectively). Our study revealed that icariin can potentially compete with coadministrated drugs for particular SLCs, which may impact the therapeutic outcome of regimens.

Interactions of the active components of Punica granatum (pomegranate) with the essential renal and hepatic human Solute Carrier transporters.

Abstract Context: Solute carrier transporters (SLCs) are membrane proteins responsible for cellular influx of various substances including many pharmaceutical agents; therefore, they largely impact on drug disposition and elimination in body. Punica granatum Linnaeus (Lythraceae), pomegranate, is a fruit with antidiabetic potential. Oleanolic acid (OA), ursolic acid (UA), and gallic acid (GA) are the major bioactive components of pomegranate. Co-administration of these compounds with other drugs could result in altered drug pharmacokinetics, possibly due to competing for transporter proteins. Objective: We investigated the interactions of these three compounds with the essential hepatic and renal SLC transporters. Materials and methods: Uptake of radiolabeled transporter model substrates was assessed in HEK293 cells over-expressing SLC transporters including the organic anion transporters (OATs), organic anion transporting polypeptides (OATPs) and organic cation transporters (OCTs), in the presence or absence of 10.0 µM UA, OA, or GA. Their IC50 values on specific SLC transporters were also evaluated using varying concentrations of the particular compound (ranging from 0.10 nM to 80.0 µM). Results: Our results demonstrated UA could significantly inhibit OAT3 and OATP2B1 uptake (IC50: 18.9 ± 8.20 µM and 11.0 ± 5.00 µM, respectively) and GA has a pronounced inhibitory effect on OATP1B3 uptake (IC50: 1.60 ± 0.60 μM). Discussion and conclusion: Our study reports the interactions of OA, UA, and GA with the essential SLC transporters. This information may contribute to elucidating the drug–drug/herb interactions involved with these three compounds and form the basis of therapeutic optimization when drugs are co-administered.

Human oligopeptide transporter 2 (PEPT2) mediates cellular uptake of polymyxins

OBJECTIVES Polymyxins are a last-line therapy to treat MDR Gram-negative bacterial infections. Nephrotoxicity is the dose-limiting factor for polymyxins and recent studies demonstrated significant accumulation of polymyxins in renal tubular cells. However, little is known about the mechanism of polymyxin uptake into these cells. Oligopeptide transporter 2 (PEPT2) is a solute carrier transporter (SLC) expressed at the apical membrane of renal proximal tubular cells and facilitates drug reabsorption in the kidney. In this study, we examined the role of PEPT2 in polymyxin uptake into renal tubular cells. METHODS We investigated the inhibitory effects of colistin and polymyxin B on the substrate uptake mediated through 15 essential SLCs in overexpressing HEK293 cells. The inhibitory potency of both polymyxins on PEPT2-mediated substrate uptake was measured. Fluorescence imaging was employed to investigate PEPT2-mediated uptake of the polymyxin fluorescent probe MIPS-9541 and a transport assay was conducted with MIPS-9541 and [(3)H]polymyxin B1. RESULTS Colistin and polymyxin B potently inhibited PEPT2-mediated [(3)H]glycyl-sarcosine uptake (IC50 11.4 ± 3.1 and 18.3 ± 4.2 μM, respectively). In contrast, they had no or only mild inhibitory effects on the transport activity of the other 14 SLCs evaluated. MIPS-9541 potently inhibited PEPT2-mediated [(3)H]glycyl-sarcosine uptake (IC50 15.9 μM) and is also a substrate of PEPT2 (Km 74.9 μM). [(3)H]polymyxin B1 was also significantly taken up by PEPT2-expressing cells (Km 87.3 μM). CONCLUSIONS Our study provides the first evidence of PEPT2-mediated uptake of polymyxins and contributes to a better understanding of the accumulation of polymyxins in renal tubular cells.

PDZK1 and NHERF1 Regulate the Function of Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) by Modulating Its Subcellular Trafficking and Stability

The human organic anion transporting polypeptide 1A2 (OATP1A2) is an important membrane protein that mediates the cellular influx of various substances including drugs. Previous studies have shown that PDZ-domain containing proteins, especially PDZK1 and NHERF1, regulate the function of related membrane transporters in other mammalian species. This study investigated the role of PDZK1 and NHERF1 in the regulation of OATP1A2 in an in vitro cell model. Transporter function and protein expression were assessed in OATP1A2-transfected HEK-293 cells that co-expressed PDZK1 or NHERF1. Substrate (estrone-3-sulfate) uptake by OATP1A2 was significantly increased to ∼1.6- (PDZK1) and ∼1.8- (NHERF1) fold of control; this was dependent on the putative PDZ-binding domain within the C-terminus of OATP1A2. The functional increase of OATP1A2 following PDZK1 or NHERF1 over-expression was associated with increased transporter expression at the plasma membrane and in the whole cell, and was reflected by an increase in the apparent maximal velocity of estrone-3-sulfate uptake (Vmax: 138.9±4.1 (PDZK1) and 181.4±16.7 (NHERF1) versus 55.5±3.2 pmol*(µg*4 min)−1 in control; P<0.01). Co-immunoprecipitation analysis indicated that the regulatory actions of PDZK1 and NHERF1 were mediated by direct interaction with OATP1A2 protein. In further experiments PDZK1 and NHERF1 modulated OATP1A2 expression by decreasing its internalization in a clathrin-dependent (but caveolin-independent) manner. Additionally, PDZK1 and NHERF1 enhanced the stability of OATP1A2 protein in HEK-293 cells. The present findings indicated that PDZK1 and NHERF1 regulate the transport function of OATP1A2 by modulating protein internalization via a clathrin-dependent pathway and by enhancing protein stability.

Human organic anion transporting polypeptide 1A2 (OATP1A2) mediates cellular uptake of all-trans-retinol in human retinal pigmented epithelial cells

Vision depends on retinoid exchange between the retinal pigment epithelium (RPE) and photoreceptors. Defects in any step of the canonical visual cycle can lead to retinal degenerations. All‐trans‐retinol (atROL) plays an important role in visual signal transduction. However, how atROL enters human RPE from the apical membrane remains unclear. This study investigated the role of human organic anion transporting polypeptide 1A2 (OATP1A2) in atROL uptake in human RPE.

Chloroquine and Hydroxychloroquine Are Novel Inhibitors of Human Organic Anion Transporting Polypeptide 1A2

Chloroquine (CQ) and hydroxychloroquine (HCQ) are widely used to treat malaria and inflammatory diseases, long-term usage of which often causes severe side effects, especially retinopathy. Solute carrier transporters (SLCs) are important proteins responsible for the cellular uptake of endogenous and exogenous substances. Inhibitors competing with transporter substrates for SLCs often results in unfavorable toxicities and unsatisfactory therapeutic outcomes. We investigated the inhibitory effect of CQ and HCQ on substrate uptake mediated through a range of important SLC transporters in overexpressing human embryonic kidney (HEK293) cells. Our data revealed that both CQ and HCQ potently inhibit the uptake activity of organic anion transporting polypeptide 1A2 (OATP1A2). We recently reported OATP1A2 to be expressed in human retinal pigment epithelium (RPE), where it mediates cellular uptake of all-trans-retinol (atROL), a key step in the classical visual cycle. In this study, we demonstrate that CQ and HCQ could markedly impair atROL uptake in OATP1A2-expressing HEK293 cells and more importantly, in primary human RPE cells. Our study shows that CQ and HCQ are novel inhibitors of OATP1A2 and significantly impair OATP1A2-mediated substrate uptake, particularly transport of atROL into the RPE. This effect may compromise the function of the classic visual cycle leading to vision impairment and contribute to the retinopathy observed clinically in patients using CQ or HCQ.

The inhibitory effects of camptothecin (CPT) and its derivatives on the substrate uptakes mediated by human solute carrier transporters (SLCs)

Abstract 1. Camptothecin (CPT) and its derivatives are potent candidate compounds in treating cancers. However, their clinical applications are largely restricted by severe toxicities. 2. The solute carrier transporters (SLCs), particularly the organic anion transporting polypeptides and organic anion/cation transporters (OATs/OCTs) are widely expressed in human key organs and responsible for the cellular influx of many substances including endogenous substrates and many clinically important drugs. Drug–drug interactions through SLCs often result in unsatisfied therapeutic outcomes and/or unexpected toxicities. 3. This study investigated the inhibitory effects of CPT and its eight derivatives on the cellular uptake of specific substrates mediated by the essential SLCs in over-expressing Human embryonic kidney 293 cells. 4. Our data revealed that CPT, 10-hydroxycamptothecin (HCPT), 10-methoxycamptothecin (MCPT) and 9-nitrocamptothecin (9NC) significantly inhibit the uptake activity of OAT3. 9NC also inhibited the substrate transport mediated by OAT1. The substrate uptakes of OAT1, OCTN1 and OCTN2 were significantly decreased in the presence of CZ112, while CPT-11 potently down-regulated the transport activity of OCT1 and OCT3. 5. In summary, our study demonstrated that CPT and its eight derivatives selectively inhibit the substrate uptakes mediated by the essential SLCs. This information contributes to understanding the localized toxicity of CPTs and provides novel molecular targets for the therapeutic optimization of CPTs in the future.

Casein Kinase 2 Is a Novel Regulator of the Human Organic Anion Transporting Polypeptide 1A2 (OATP1A2) Trafficking

Human organic anion transporting polypeptides (OATPs) mediate the influx of many important drugs into cells. Casein kinase 2 (CK2) is a critical protein kinase that phosphorylates >300 protein substrates and is dysregulated in a number of disease states. Among the CK2 substrates are several transporters, although whether this includes human OATPs has not been evaluated. The current study was undertaken to evaluate the regulation of human OATP1A2 by CK2. HEK-239T cells in which OATP1A2 was overexpressed were treated with CK2 specific inhibitors or transfected with CK2 specific siRNA, and the activity, expression, and subcellular trafficking of OATP1A2 was evaluated. CK2 inhibition decreased the uptake of the prototypic OATP1A2 substrate estrone-3-sulfate (E3S). Kinetic studies revealed that this was due to a decrease in the maximum velocity (Vmax) of E3S uptake, while the Michaelis constant was unchanged. The cell surface expression, but not the total cellular expression of OATP1A2, was impaired by CK2 inhibition and knockdown of the catalytic α-subunits of CK2. CK2 inhibition decreased the internalization of OATP1A2 via a clathrin-dependent pathway, decreased OATP1A2 recycling, and likely impaired OATP1A2 targeting to the cell surface. Consistent with these findings, CK2 inhibition also disrupted the colocalization of OATP1A2 and Rab GTPase (Rab)4-, Rab8-, and Rab9-positive endosomal and secretory vesicles. Taken together, CK2 has emerged as a novel regulator of the subcellular trafficking and stability of OATP1A2. Because OATP1A2 transports many molecules of physiological and pharmacological importance, the present data may inform drug selection in patients with diseases in which CK2 and OATP1A2 are dysregulated.