Caco-2细胞中药物转运蛋白的mRNA表达及活力评价

目的 系统研究Caco-2细胞中各药物转运蛋白的mRNA表达水平及转运活力,对比其与人正常肠道中药物转运蛋白表达的差异。方法 实时荧光定量PCR（qRT-PCR）方法测定Caco-2细胞中人肠道相关转运蛋白MDR1、BCRP、MRP2、OATP1A2、OATP2B1和PEPT1的表达水平;将Caco-2细胞接种于Transwell板内培养21 d并给予不同药物转运蛋白的底物及抑制剂,评价Caco-2细胞中相关转运蛋白的转运活性。结果 qRT-PCR结果表明,药物转运蛋白MDR1、MRP2、BCRP和OATP2B1在Caco-2细胞中均有相对高的表达,表达量的顺序为：MDR1 > MRP2 > OATP2B1 > BCRP,在正常人肠道表达量顺序为BCRP > MDR1 > MRP2 > OATP2B1;转运蛋白活力评价表明,各药物转运蛋白的活力测试结果均为阳性,验证了基因的表达水平结果。结论 Caco-2细胞中表达正常人体肠道表达的部分药物转运蛋白（MDR1、MRP2、BCRP和OATP2B1）,表达水平与正常人体肠道中大致相当,但也存在一定差异。     

Insulin stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1 in the human intestinal cell line Caco-2

Organic anion transporting polypeptide 2B1 (OATP2B1) is the major uptake transporter in the intestine, and transports various clinically used therapeutic agents. Insulin acts through the insulin receptor in targeted cells, and Rab8A is one of the insulin signaling pathways. The small intestine in humans also expresses insulin receptor and Rab8A. It has been reported that insulin stimulates peptide transporter 1 (PEPT1) expression at the apical membrane and increases uptake of PEPT1 substrates in small intestine epithelial model cells (Caco-2 cells). However, the effect of insulin on OATP2B1 in the small intestine has not been fully investigated. We found that Rab8A was associated with OATP2B1-mediated estrone-3-sulfate (E3S) uptake. Insulin stimulated the uptake of E3S by Caco-2 cells and the enhancement was sustained for 120 min. The Vmax value of E3S uptake significantly increased upon insulin exposure. Caco-2 cells treated with insulin showed increased OATP2B1 expression at the cell surface. The apical-to-basal transport of E3S was also increased by insulin. The increase of E3S transport was inhibited by the cold condition (4 °C) or the OATP2B1 inhibitor, taurocholate. These results indicate that insulin acts on the small intestine to increase OATP2B1-mediated absorption.     

Pharmacokinetic analysis of transcellular transport of levofloxacin across LLC-PK1 and Caco-2 cell monolayers

To characterize the membrane transport responsible for the renal excretion and intestinal absorption of levofloxacin, we performed pharmacokinetic analysis of transcellular transport across LLC-PK(1) and Caco-2 cell monolayers. Transcellular transport of levofloxacin in LLC-PK(1) cells was greater in the basolateral-to-apical direction than in the opposite direction. Pharmacokinetic analysis indicated that basolateral uptake was the direction-determining step for the transcellular transport of levofloxacin in LLC-PK(1) cells. The apical efflux clearance of levofloxacin in LLC-PK(1) cells was increased at the medium pH 6 as compared with at pH 8, suggesting that membrane transport characteristics of levofloxacin are apparently similar to those of a prototypical organic cation, tetraethylammonium. On the other hand, transcellular transport of levofloxacin in Caco-2 cells was only slightly greater in the basolateral-to-apical direction than in the opposite direction. The apical efflux clearance of levofloxacin in Caco-2 cells was greater than basolateral efflux clearance, and apical influx clearance was greater than any other membrane transport clearance. In addition, the apical uptake of levofloxacin as well as quinidine in Caco-2 cells was inhibited significantly by nicotine and imipramine. The findings indicated that some transporters are responsible not only for the efflux but also for the influx of levofloxacin at the apical membrane of Caco-2 cells.     

The apical uptake transporter of levofloxacin is distinct from the peptide transporter in human intestinal epithelial Caco-2 cells

The aim of this study was to investigate the involvement of the peptide transporter for absorption of levofloxacin in Caco-2 cells. To evaluate the activity of apical and basolateral peptide transport, we first performed pharmacokinetic analysis of transcellular transport of glycylsarcosine (Gly-Sar) in cell monolayers grown on porous membrane filters. Transcellular transport of Gly-Sar at the medium pH 6 was greater in the apical-to-basolateral direction than in the opposite direction. Influx clearance of Gly-Sar at the apical membrane was much greater than basolateral influx and efflux clearance, indicating that the apical peptide transporter plays an important role in directional transcellular transport of the dipeptide across Caco-2 cell monolayers. We then evaluated the effect of various compounds on the uptake of Gly-Sar and levofloxacin at the apical membrane of Caco-2 cells. The apical uptake of [3H]Gly-Sar was significantly inhibited by Ala-Ala, Gly-Sar, and also levofloxacin, whereas that of [14C]levofloxacin was not inhibited by Ala-Ala and Gly-Sar. On the other hand, the apical uptake of [14C]levofloxacin was inhibited by nicotine, enalapril, fexofenadine, and L-carnitine. These findings indicated that the apical uptake transporter of levofloxacin is distinct from the peptide transporter in Caco-2 cells.     

Vectorial transport of fexofenadine across Caco-2 cells: involvement of apical uptake and basolateral efflux transporters

Fexofenadine is a nonsedative antihistamine that exhibits good oral bioavailability despite its zwitterionic chemical structure and efflux by P-gp. Evidence exists that multiple uptake and efflux transporters play a role in hepatic disposition of fexofenadine. However, the roles of specific transporters and their interrelationship in intestinal absorption of this drug are unclear. This study was designed to elucidate vectorial absorptive transport of fexofenadine across Caco-2 cells involving specific apical uptake and efflux transporters as well as basolateral efflux transporters. Studies with cellular models expressing single transporters showed that OATP2B1 expression stimulated uptake of fexofenadine at pH 6.0. Apical uptake of fexofenadine into Caco-2 cells was decreased by 45% by pretreatment with estrone 3-sulfate, an OATP inhibitor, at pH 6.0 but not at pH 7.4, indicating that OATP2B1 mediates apical uptake of fexofenadine into these cells. Examination of fexofenadine efflux from preloaded Caco-2 cells in the presence or absence of (i) the MRP inhibitor MK-571 and (ii) the P-gp inhibitor GW918 showed that apical efflux is predominantly mediated by P-gp, with a small contribution by MRP2, whereas basolateral efflux is predominantly mediated by MRP3. These results also showed that while OSTαβ is functionally active in the basolateral membrane of Caco-2 cells, it does not play a role in the export of fexofenadine. MK-571 decreased the absorptive transport of fexofenadine by 17%. However, the decrease in absorptive transport by MK-571 was 42% when P-gp was inhibited by GW918. The results provide a novel insight into a vectorial transport system mainly consisting of apical OATP2B1 and basolateral MRP3 that may play an important role in delivering hydrophilic anionic and zwitterionic drugs such as pravastatin and fexofenadine into systemic circulation upon oral administration.     

Utility of P-glycoprotein and organic cation transporter 1 double-transfected LLC-PK1 cells for studying the interaction of YM155 monobromide, novel small-molecule survivin suppressant, with P-glycoprotein

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), a novel small molecule that downregulates survivin and exhibits potent antitumor activity, is hydrophilic and cationic. Although previous studies have shown that influx transporters play important roles in the uptake of YM155 into hepatocytes and possibly into cancer cells, efflux transporters have yet to be investigated. In this study, we assessed the interaction of YM155 with P-glycoprotein [multidrug resistance 1 (MDR1)/ATP-binding cassette B1] using two kinds of transcellular transport systems: Caco-2 and MDR1-expressing LLC-PK1 cells (LLC-MDR1). We also used a newly established LLC-OCT1/MDR1 cell line, which expresses basal YM155 uptake transporter organic cation transporter1 (OCT1) and apical MDR1. Direct interaction between YM155 and MDR1 and other efflux transporters was evaluated using transporter-expressing membrane vesicles. A bidirectional transporter assay using Caco-2 and LLC-MDR1 cells showed low permeability and no vectorial transport of YM155, suggesting that YM155 is not a substrate of MDR1. However, vectorial transport across LLC-OCT1/MDR1 cells was identified, which was inhibited by the MDR1 inhibitor cyclosporine A, clearly indicating that YM155 is in fact a substrate of MDR1. Insufficient expression of basal uptake transporter of YM155 in Caco-2 and LLC-MDR1 might have confounded conclusions regarding YM155 and MDR1. Using the transporter-expressing vesicles, MDR1-mediated transport was most significantly involved in YM155 transport among the efflux transporters examined. In conclusion, these findings suggest that YM155 is a substrate of MDR1, and that MDR1 may play an important role in the pharmacokinetics of YM155. Transcellular assays lacking basal uptake transporters may be inaccurate in the assessment of hydrophilic compounds that have poor membrane permeability by passive diffusion.     

Polarized expression of drug transporters in differentiated human hepatoma HepaRG cells

The HepaRG cell line is a well-differentiated human hepatoma cell line proposed as a surrogate for human hepatocytes, especially for hepatic detoxification studies. Polarized status of drug transporters, i.e., their coordinated location at sinusoidal or canalicular membranes, which represents a key hallmark of hepato-biliary drug transport, remains however incompletely documented in HepaRG cells. The present study was therefore designed to analyze transporter location in HepaRG cells, which exhibit mRNA expressions of most of hepatic transporters. HepaRG cells were demonstrated, through immunofluorescence staining, to express several drug transporters at their sinusoidal pole, especially the influx transporters organic anion transporting polypeptide (OATP) 1B1, OATP2B1 and organic cation transporter (OCT) 1 and the efflux transporter multidrug resistance-associated protein (MRP) 3. In addition, the efflux transporters P-glycoprotein and MRP2 were detected at the canalicular pole of HepaRG cells. Moreover, saturable uptake of reference substrates for the sinusoidal transporters sodium-taurocholate cotransporting polypeptide, OATPs and OCT1 and canalicular secretion of reference substrates for the efflux transporters bile salt export pump and MRP2 were observed. This polarized and functional expression of various sinusoidal and canalicular transporters in HepaRG cells highlights the interest of using these hepatoma cells in xenobiotic transport studies.     

pH-sensitive interaction of HMG-CoA reductase inhibitors (statins) with organic anion transporting polypeptide 2B1

The human organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) is ubiquitously expressed and may play an important role in the disposition of xenobiotics. The present study aimed to examine the role of OATP2B1 in the intestinal absorption and tissue uptake of 3-hydroxy-3-methylglutaryl-Coenzyme A (HMG-CoA) reductase inhibitors (statins). We first investigated the functional affinity of statins to the transporter as a function of extracellular pH, using OATP2B1-transfeced HEK293 cells. The results indicate that OATP2B1-mediated transport is significant for rosuvastatin, fluvastatin and atorvastatin, at neutral pH. However, OATP2B1 showed broader substrate specificity as well as enhanced transporter activity at acidic pH. Furthermore, uptake at acidic pH was diminished in the presence of proton ionophore, suggesting proton gradient as the driving force for OATP2B1 activity. Notably, passive transport rates are predominant or comparable to active transport rates for statins, except for rosuvastatin and fluvastatin. Second, we studied the effect of OATP modulators on statin uptake. At pH 6.0, OATP2B1-mediated transport of atorvastatin and cerivastatin was not inhibitable, while rosuvastatin transport was inhibited by E-3-S, rifamycin SV and cyclosporine with IC(50) values of 19.7 ± 3.3 μM, 0.53 ± 0.2 μM and 2.2 ± 0.4 μM, respectively. Rifamycin SV inhibited OATP2B1-mediated transport of E-3-S and rosuvastatin with similar IC(50) values at pH 6.0 and 7.4, suggesting that the inhibitor affinity is not pH-dependent. Finally, we noted that OATP2B1-mediated transport of E-3-S, but not rosuvastatin, is pH sensitive in intestinal epithelial (Caco-2) cells. However, uptake of E-3-S and rosuvastatin by Caco-2 cells was diminished in the presence of proton ionophore. The present results indicate that OATP2B1 may be involved in the tissue uptake of rosuvastatin and fluvastatin, while OATP2B1 may play a significant role in the intestinal absorption of several statins due to their transporter affinity at acidic pH.     

Impact of OATP transporters on pharmacokinetics

Membrane transporters are now recognized as important determinants of the transmembrane passage of drugs. Organic anion transporting polypeptides (OATP) form a family of influx transporters expressed in various tissues important for pharmacokinetics. Of the 11 human OATP transporters, OATP1B1, OATP1B3 and OATP2B1 are expressed on the sinusoidal membrane of hepatocytes and can facilitate the liver uptake of their substrate drugs. OATP1A2 is expressed on the luminal membrane of small intestinal enterocytes and at the blood-brain barrier, potentially mediating drug transport at these sites. Several clinically used drugs have been identified as substrates of OATP transporters (e.g. many statins are substrates of OATP1B1). Some drugs may inhibit OATP transporters (e.g. cyclosporine) causing pharmacokinetic drug–drug interactions. Moreover, genetic variability in genes encoding OATP transporters can result in marked inter-individual differences in pharmacokinetics. For example, a single nucleotide polymorphism (c.521T > C, p.Val174Ala) in the SLCO1B1 gene encoding OATP1B1 decreases the ability of OATP1B1 to transport active simvastatin acid from portal circulation into the liver, resulting in markedly increased plasma concentrations of simvastatin acid and an enhanced risk of simvastatin-induced myopathy. SLCO1B1 polymorphism also affects the pharmacokinetics of many other, but not all (fluvastatin), statins and that of the antidiabetic drug repaglinide, the antihistamine fexofenadine and the endothelin A receptor antagonist atrasentan. This review compiles the current knowledge about the expression and function of human OATP transporters, their substrate and inhibitor specificities, as well as pharmacogenetics.     

Involvement of transporters in the hepatic uptake and biliary excretion of valsartan, a selective antagonist of the angiotensin II AT1-receptor, in humans.

Valsartan is a highly selective angiotensin II AT1-receptor antagonist for the treatment of hypertension. Valsartan is mainly excreted into the bile in unchanged form. Because valsartan has an anionic carboxyl group, we hypothesized that a series of organic anion transporters could be involved in its hepatic clearance. In this study, to identify transporters that mediate the hepatic uptake and biliary excretion of valsartan and estimate the contribution of each transporter to the overall hepatic uptake and efflux, we characterized its transport using transporter-expressing systems, human cryopreserved hepatocytes, and Mrp2-deficient Eisai hyperbilirubinemic rats (EHBRs). Valsartan was significantly taken up into organic anion-transporting polypeptide (OATP) 1B1 (OATP2/OATP-C)- and OATP1B3 (OATP8)-expressing HEK293 cells. We also observed saturable uptake into human hepatocytes. Based on our estimation, the relative contribution of OATP1B1 to the uptake of valsartan in human hepatocytes depends on the batch, ranging from 20 to 70%. Regarding efflux transporters, the ratio of basal-to-apical transcellular transport of valsartan to that in the opposite direction in OATP1B1/MRP2 (multidrug resistance-associated protein 2) double transfected cells was the highest among the three kinds of double transfectants, OATP1B1/MRP2, OATP1B1/multi-drug resistance 1, and OATP1B1/breast cancer resistance protein-expressing MDCKII cells. We observed saturable ATP-dependent transport into membrane vesicles expressing human MRP2. We also found that the elimination of intravenously administered valsartan from plasma was markedly delayed, and the biliary excretion was severely impaired in EHBR compared with normal Sprague-Dawley rats. These results suggest that OATP1B1 and OATP1B3 as the uptake transporters and MRP2 as the efflux transporter are responsible for the efficient hepatobiliary transport of valsartan.     

Interaction of sirolimus and everolimus with hepatic and intestinal organic anion-transporting polypeptide transporters

The goal of this study was to assess the interaction of the mTOR inhibitors (ImTORs) sirolimus and everolimus with the human organic anion-transporting polypeptides (OATPs) expressed in hepatocytes and enterocytes by conducting uptake experiments using (i) transfected HEK293T cells, (ii) the hepatocyte-like HepaRG cell line and (iii) the enterocyte-like Caco-2 cell line. Sirolimus and everolimus inhibited in a dose-dependent manner the uptake of [3H]-estrone sulphate by OATP1A2 and OATP1B1 and that of mycophenolic acid 7-O-glucuronide (MPAG) by OATP1B3. ImTOR apparent 50% inhibitory concentrations (IC??) for OATPs were 11.9 μM (OATP1A2), 9.8 μM (OATP1B1) and 1.3 μM (OATP1B3) for sirolimus and 4.2 μM (OATP1A2), 4.1 μM (OATP1B1) and 4.3 μM (OATP1B3) for everolimus. No transport of sirolimus or everolimus by OATP1A2, OATP1B1 or OATP1B3 was observed in HEK-transfected cells and the OAT/OATP/MRP chemical inhibitor probenecid did not significantly decrease the uptake of sirolimus and everolimus in HepaRG and Caco-2 cells, but tended to increase their intracellular accumulation presumably through efflux inhibition. In conclusion, our data suggest that the major OATP transporters expressed in the liver and the intestine do not contribute to the pharmacokinetics of sirolimus and everolimus. However, ImTORs are inhibitors of these transporters.     

Regulation of drug transporter expression by oncostatin M in human hepatocytes

The cytokine oncostatin M (OSM) is a member of the interleukin (IL)-6 family, known to down-regulate expression of drug metabolizing cytochromes P-450 in human hepatocytes. The present study was designed to determine whether OSM may also impair expression of sinusoidal and canalicular drug transporters, which constitute important determinants of drug hepatic clearance. Exposure of primary human hepatocytes to OSM down-regulated mRNA levels of major sinusoidal solute carrier (SLC) influx transporters, including sodium-taurocholate co-transporting polypeptide (NTCP), organic anion transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1, organic cation transporter 1 and organic anion transporter 2. OSM also repressed mRNA expressions of ATP binding cassette (ABC) efflux transporters such as multidrug resistance protein (MRP) 2/ABCC2 and breast cancer resistance protein/ABCG2, without however impairing those of multidrug resistance gene 1/P-glycoprotein/ABCB1, MRP3/ABCC3, MRP4/ABCC4 and bile salt export pump/ABCB11. The cytokine concomitantly reduced NTCP, OATP1B1, OATP2B1 and ABCG2 protein expression and NTCP and OATP transport activities. OSM effects towards transporters were found to be dose-dependent and highly correlated with those of IL-6, but not with those of other inflammatory cytokines such as tumor necrosis factor-α or interferon-γ. In addition, OSM-mediated repression of some transporters such as NTCP, OATP1B1 and OATP2B1, was counteracted by knocking-down expression of the type II OSM receptor subunits through siRNA transfection. This OSM-mediated down-regulation of drug SLC transporters and ABCG2 in human hepatocytes may contribute to alterations of pharmacokinetics in patients suffering from diseases associated with increased production of OSM.     

Role of Uptake Transporters OAT4, OATP2A1, and OATP1A2 in Human Placental Bio-disposition of Pravastatin

Pravastatin is currently under evaluation for prevention of preeclampsia. Factors contributing to placental disposition of pravastatin are important in assessment of potential undesirable fetal effects. The purpose of this study was to identify the uptake transporters that contribute to the placental disposition of pravastatin. Our data revealed the expression of organic anion transporting polypeptide 1A2 (OATP1A2) and OATP2A1 in the apical, and OATP2B1 and OATP5A1 in the basolateral membranes of the placenta, while organic anion transporter 4 (OAT4) exhibited higher expression in basolateral membrane but was detected in both membranes. Preloading placental membrane vesicles with glutarate increased the uptake of pravastatin suggesting involvement of glutarate-dependent transporters such as OAT4. In the HEK293 cells overexpressing individual uptake transporters, OATP2A1, OATP1A2 and OAT4 were determined to accept pravastatin as a substrate at physiological pH, while the uptake of pravastatin by OATP2B1 (known to interact with pravastatin at acidic pH) and OATP5A1 was not detected at pH 7.4. These findings led us to propose that OATP1A2 and OATP2A1 are responsible for the placental uptake of pravastatin from the maternal circulation, while OAT4 mediates the passage of the drug across placental basolateral membrane in the fetal-to-maternal direction.     

Simulations of the Nonlinear Dose Dependence for Substrates of Influx and Efflux Transporters in the Human Intestine

The purpose of this study was to develop simulation and modeling methods for the evaluation of pharmacokinetics when intestinal influx and efflux transporters are involved in gastrointestinal absorption. The advanced compartmental absorption and transit (ACAT) model as part of the computer program GastroPlus™ was used to simulate the absorption and pharmacokinetics of valacyclovir, gabapentin, and talinolol. Each of these drugs is a substrate for an influx or efflux transporter and all show nonlinear dose dependence within the normal therapeutic range. These simulations incorporated the experimentally derived gastrointestinal distributions of transporter expression levels for oligopeptide transporters PepT1 and HPT1 (valacyclovir); System L-amino acid transporter LAT2 and organic cation transporter OCTN1 (gabapentin); and organic anion transporter (OATP1A2) and P-glycoprotein (talinolol). By assuming a uniform distribution of oligopeptide transporter and by application of the in vitro K_m value for valacyclovir, the simulations accurately reproduced the experimental nonlinear dose dependence. For gabapentin, LAT2 distribution produced simulation results that were much more accurate than OCTN1 distributions. For talinolol, an influx transporter distribution for OATP1A2 and the efflux transporter P-glycoprotein distributed with increasing expression in the distal small intestine produced the best results. The physiological characteristics of the small and large intestines used in the ACAT model were able to accurately account for the positional and temporal changes in concentration and carrier-mediated transport of the three drugs included in this study. The ACAT model reproduced the nonlinear dose dependence for each of these drugs.Key words: expression, intestine, saturation, simulation, transporter     

Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells.

Human organic anion transporting polypeptide OATP-B (OATP2B1) is a pH-sensitive transporter expressed in the apical membranes of small intestinal epithelial cells. In this study, we have examined the contribution of OATP-B to the uptake of [3H]estrone-3-sulfate in Caco-2 cells in comparison with those of its homologs OATP-D (OATP3A1) and OATP-E (OATP4A1). Immunocytochemical study revealed that OATP-B is expressed in the apical membranes of Caco-2 cells. The uptake of [3H]estrone-3-sulfate by Caco-2 cells was Na+-independent and inhibited by several organic anions. It showed biphasic saturation kinetics with Km values of 1.81 microM and 1.40 mM. The uptake of [3H]estrone-3-sulfate by human embryonic kidney (HEK) 293 cells stably expressing OATP-B (HEK293/OATP-B) was also Na+-independent and inhibited by several organic anions. The Km value for estrone-3-sulfate uptake by OATP-B (1.56 microM) was close to that for the high-affinity component observed in Caco-2 cells. The mRNA expression level of OATP-B was higher than that of OATP-D or OATP-E in Caco-2 cells and in human jejunum biopsies from healthy volunteers. The values of [3H]estrone-3-sulfate uptake normalized to OATP-B mRNA expression were similar in Caco-2 cells and HEK293/OATP-B cells. The specific activity of OATP-B per mRNA expression was much higher than that of OATP-D and OATP-E. [3H]Estrone-3-sulfate uptake by membrane vesicles prepared from HEK293/OATP-B cells exhibited an overshoot phenomenon in the presence of an inwardly directed H+ gradient, suggesting that an H+ gradient is the driving force of estrone-3-sulfate transport by OATP-B. These results suggest that OATP-B is predominantly responsible for the apical uptake of estrone-3-sulfate in Caco-2 cells.     

Directional trans-epithelial transport of organic anions in porcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2

The transcellular transport of many compounds, which cannot readily cross the lipid bilayer, is mediated by drug uptake and efflux transporters. Human OATP1B1 and MRP2 have been implicated in the hepato-biliary transport of many endogenous and exogenous compounds. Here, we have established epithelial porcine kidney LLC-PK1 derived cell lines, that express both transporters in a polarized fashion, as a model to predict hepato-biliary transport. Immunological identification of OATP1B1 in the recombinant cell lines was greatly facilitated by its C-terminal tagging with a peptide sequence derived from hemagglutinin (HA) avoiding the generation of OATP1B1 specific antibodies. Importantly, the tag did not interfere with the functionality of the transporter. Compared to LLC-PK1 cells and cells which expressed only OATP1B1, the cell line that co-expressed MRP2 and OATP1B1 displayed high directional basolateral-to-apical transport of 17 beta-estradiol-17 beta-glucuronide and estrone-3-sulfate. Dehydroepiandrosterone sulfate already displayed a significant basolateral-to-apical transport in the parental cell line, which was further stimulated upon expression of both transporters. Transcellular flux of all steroid conjugates in the opposite direction (apical-to-basolateral) was much lower. By employing this cellular model we were able to demonstrate for the first time that OATP1B1 together with MRP2 mediates the trans-cellular transport of rifampicin. It is anticipated that the models established herein will greatly facilitate the identification of transporters involved in the disposition of novel drug candidates.     

Multiple human isoforms of drug transporters contribute to the hepatic and renal transport of olmesartan, a selective antagonist of the angiotensin II AT1-receptor.

Olmesartan, a novel angiotensin II AT1-receptor antagonist, is excreted into both bile and urine, with minimal metabolism. Because olmesartan is a hydrophilic anionic compound, some transporters could be involved in its hepatic and renal clearance. In this study, we characterized the role of human drug transporters in the pharmacokinetics of olmesartan and determined the contribution of each transporter to the overall clearance of olmesartan. Olmesartan was significantly taken up into human embryonic kidney 293 cells expressing organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, organic anion transporter (OAT) 1, and OAT3. We also observed its saturable uptake into human hepatocytes and kidney slices. Estimated from the relative activity factor method and application of specific inhibitors, the relative contributions of OATP1B1 and OATP1B3 to the uptake of olmesartan in human hepatocytes were almost the same, whereas OAT3 was predominantly involved in its uptake in kidney slices. The vectorial transport of olmesartan was observed in OATP1B1/multidrug resistance-associated protein (MRP) 2 double transfectants, but not in OATP1B1/multidrug resistance (MDR) 1 and OATP1B1/breast cancer resistance protein (BCRP) transfectants. ATP-dependent transport into membrane vesicles expressing human MRP2 and MRP4 was clearly observed, with K(m) values of 14.9 and 26.2 microM, respectively, whereas the urinary excretion of olmesartan in Mrp4-knockout mice was not different from that of control mice. We also investigated the transcellular transport of olmesartan medoxomil, a prodrug of olmesartan. Vectorial basal-to-apical transport was observed in OATP1B1/MRP2, OATP1B1/MDR1 double, and OATP1B1/BCRP double transfectants, suggesting the possible involvement of MRP2, MDR1, and BCRP in the limit of intestinal absorption of olmesartan medoxomil. From these results, we suggest that multiple transporters make a significant contribution to the pharmacokinetics of olmesartan and its prodrug.     

Variability in mRNA expression of ABC- and SLC-transporters in human intestinal cells: comparison between human segments and Caco-2 cells.

The Caco-2 cell monolayer model is widely used as a tool for evaluating human intestinal permeability and interaction with transporters. Therefore, we determined mRNA levels for 15 of the most frequently studied uptake and efflux transporters (MDR1, MRP2-3, BCRP, OCTN2, PepT1, OATP-B, OATP8, OCT1-3, OAT1-3, MCT1) using real-time PCR in Caco-2 cells and in human jejunum and colon. The expression levels in the Caco-2 cells did not significantly vary between different passages (p29-43) and batches for any of the genes measured. However, levels increased with culture time (1-5 weeks) for PepT1, MDR1, MRP2, OATP-B and BCRP. The general rank order of the gene expression levels in Caco-2 cells was established as follows: MRP2>OATP-B>PepT1>MDR1>MCT1 approximately MRP3 approximately BCRP approximately OCTN2>OCT3>OCT1>OAT2. Four genes were absent: OATP8, OCT2, OAT1, and OAT3. Ranking of 11 expressed genes showed a significant correlation between human jejunum and 2-5-week-old Caco-2 cells. The expression profile in colon was, however, very different compared to both Caco-2 cells and jejunum. We conclude that the Caco-2 cells in our hands express similar transporters as the human jejunum, but are different from colon, indicating their usefulness for obtaining small intestinal transport data. In addition, we also suggest that cells with a well-defined range of culture ages should be used to minimize variability in data from experiments and even erroneous conclusions.     

Oral drug delivery utilizing intestinal OATP transporters

Transporters play important roles in tissue distribution and urinary- and biliary-excretion of drugs and transporter molecules involved in those processes have been elucidated well. Furthermore, an involvement of efflux transporters such as P-glycoproteins, multidrug resistance associated protein 2, and breast cancer resistance protein as the intestinal absorption barrier and/or intestinal luminal secretion mechanisms has been demonstrated. However, although there are many suggestions for the contribution of uptake/influx transporters in intestinal absorption of drugs, information on the transporter molecules responsible for the intestinal absorptive process is limited. Among them, most studied absorptive drug transporter is peptide transporter PEPT1. However, utilization of PEPT1 for oral delivery of drugs may not be high due to the chemical structural requirement of PEPT1 limited to peptide-mimetics. Recently, organic anion transporting polypeptide (OATP) family such as OATP1A2 and OATP2B1 has been suggested to mediate intestinal absorption of several drugs. Since OATPs exhibit species difference in expressed tissues and functional properties between human and animals, human studies are essential to clarify the intestinal absorption mechanisms of drugs via OATPs. Recent pharmacogenomic studies demonstrated that OATP2B1 is involved in the drug absorption in human. In addition, information of drug-juice interaction in the intestine also uncovered the contribution of OATP1A2 and OATP2B1 in drug absorption. Since OATP1A2 and OATP2B1 exhibit broader substrate selectivity compared with PEPT1, their potential to be applied for oral delivery should be high. In this review, current understanding of characteristics and contribution as the absorptive transporters of OATPs in small intestine in human is described. Now, it is getting clearer that OATPs have significant roles in intestinal absorption of drugs, therefore, there are higher possibility to utilize OATPs as the tools for oral delivery.     

Pharmaceutical excipients influence the function of human uptake transporting proteins

Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na(+)/taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E(3)S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17β-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC(50) < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC(50) values of 0.05% (TA) and 0.14% (E(3)S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.