Substrate Of OATP1B3 Research Articles

Abstract 882: Functional investigation of a novel cancer specific organic anion transporting polypeptide 1B3 variant 1 (OATP1B3 V1) in colon and pancreatic cancer.

Abstract OATP1B3 is a member of the Organic Anion Transporting Polypeptide (OATP) superfamily that mediates the uptake of numerous endogenous and xenobiotic compounds. Initially, OATP1B3 was reported to be expressed exclusively in the normal hepatocytes. Subsequent studies have reported that OATP1B3 is frequently expressed in multiple types of cancer tissues derived from gastrointestinal tract, pancreas, prostate, lung and breast. Some reports have also suggested that OATP1B3 may be associated with differing clinical outcomes. However, so far the biological functions of OATP1B3 in cancer have not been elucidated. In this study, we confirmed that colon and pancreatic cancer cells express variant forms of OATP1B3, different from OATP1B3 wildtype (WT) expressed in normal liver. OATP1B3 variant 1 (V1) is the most prevalent form among the variants containing the alternate exon 2a, instead of exons 1 and 2 as in OATP1B3 WT. The translated protein sequence of OATP1B3 V1 is similar to OATP1B3 WT except lacking the first 28 amino acids at the N-terminus. OATP1B3 V1 has a limited transport activity toward cholecystokin-8 (CCK-8, a prototype OATP1B3 substrate) in contrast to OATP1B3 WT which showed a markedly efficient uptake of CCK-8. Consistent with these results, OATP1B3 V1 was localized mainly in the cytoplasm with a much lower extent of trafficking to the surface membrane compared to OATP1B3 WT. In summary, our results demonstrate that colon and pancreatic cancer cells express variant forms of OATP1B3 with a limited transport activity and different subcellular localization compared to OATP1B3 WT. These observed differences at molecular and functional levels will be important considerations for further investigations of the biological and clinical significance of OATP1B3 expression in cancer. Citation Format: Nilay Thakkar, Kyungbo Kim, Eun Ryoung Jang, Songhee Han, Kyunghwa Kim, Donghern Kim, Nipun Merchant, A. Craig Lockhart, Wooin Lee. Functional investigation of a novel cancer specific organic anion transporting polypeptide 1B3 variant 1 (OATP1B3 V1) in colon and pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 882. doi:10.1158/1538-7445.AM2013-882

Abstract 812: Identification of novel anticancer drugs as substrates of organic anion transporting polypeptide 1B3 using a cell viability assay

Abstract Organic anion transporting polypeptide 1B3 (OATP1B3) belongs to a superfamily of transport proteins that mediate the uptake of a wide range of endogenous and exogenous compounds into cells. Under normal conditions OATP1B3 is selectively expressed at the basolateral membrane of human hepatocytes. Recently, OATP1B3 expression has been detected in a variety of different cancers and because of its multispecificity OATP1B3 is thought to be a potential therapeutic target to enhance drug delivery into cancer cells. Thus far, only a few anticancer drugs have been identified as substrates of OATP1B3 including docetaxel, paclitaxel, methotrexate and imatinib. Given that OATP1B3 can potentially be used as a target for anticancer drug delivery we need to identify additional anticancer drug substrates of OATP1B3. Therefore, we aimed at establishing a cell based high throughput assay to screen the NCI approved oncology drug set for novel cytotoxic substrates of OATP1B3. Using a luminescent cell viability assay, we measured the viability of wild-type and OATP1B3-expressing Chinese Hamster Ovary (CHO) cells in the absence or presence of the anticancer drugs. After we identified positive hits we further characterized them with respect to concentration dependencies. We identified etoposide, bleomycin, oxaliplatin and plicamycin as compounds that kill OATP1B3-expressing CHO cells at much lower concentrations than wild-type CHO cells suggesting that these compounds are substrates of OATP1B3. Using secondary assays we confirmed that etoposide is a substrate of OATP1B3 and we are currently testing the remaining compounds for direct uptake by OATP1B3. In conclusion, this cell-based viability assay is a good tool to identify novel cytotoxic OATP1B3 substrates that can potentially be used to treat OATP1B3-expressing cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 812. doi:1538-7445.AM2012-812

Establish a cell viability assay to identify novel cytotoxic Organic Anion Transporting Polypeptide 1B3 substrates

Organic anion transporting polypeptides (OATPs) mediate the uptake of numerous structurally diverse compounds. The normally liver‐specific OATP1B3 is also expressed in different cancers, suggesting that it can be a therapeutic target for anticancer drug delivery. Because OATP1B3 also transports chemotherapy drugs, we established a cell based assay to screen for novel cytotoxic OATP1B3 substrates. We screened a library of Kansas plant extracts to isolate and chemically identify novel cytotoxic compounds that could be developed as lead compounds to treat OATP1B3‐expressing cancers. In this study, we used Chinese Hamster Ovary (CHO) cells that stably express OATP1B3 and measured cell viability of wild‐type and OATP1B3‐expressing CHO cells in the presence of Kansas plant extracts of different polarities using a luminescent cell viability assay. We identified the butanol fractions of Rhus aromatica (V345) and Rhus glabra (V585) that kill OATP1B3‐expressing CHO cells at much lower concentrations than wild‐type CHO cells, suggesting that these extracts contain cytotoxic OATP1B3‐substrates. We will further sub‐fractionate these extracts to isolate and chemically identify the cytotoxic natural products. In conclusion, this cell‐based viability assay might be a good tool to identify potential cytotoxic OATP1B3 substrates that can be delivered into OATP1B3‐expressing cancers.

Interaction of Three Regiospecific Amino Acid Residues Is Required for OATP1B1 Gain of OATP1B3 Substrate Specificity

The human organic anion-transporting polypeptides OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) are liver-enriched membrane transporters of major importance to hepatic uptake of numerous endogenous compounds, including bile acids, steroid conjugates, hormones, and drugs, including the 3-hydroxy-3-methylglutaryl Co-A reductase inhibitor (statin) family of cholesterol-lowering compounds. Despite their remarkable substrate overlap, there are notable exceptions: in particular, the gastrointestinal peptide hormone cholecystokinin-8 (CCK-8) is a high affinity substrate for OATP1B3 but not OATP1B1. We utilized homologous recombination of linear DNA by E. coli to generate a library of cDNA containing monomer size chimeric OATP1B1-1B3 and OATP1B3-1B1 transporters with randomly distributed chimeric junctions to identify three discrete regions of the transporter involved in conferring CCK-8 transport activity. Site-directed mutagenesis of three key residues in OATP1B1 transmembrane helices 1 and 10, and extracellular loop 6, to the corresponding residues in OATP1B3, resulted in a gain of CCK-8 transport by OATP1B1. The residues appear specific to CCK-8, as the mutations did not affect transport of the shared OATP1B substrate atorvastatin or the OATP1B1-specific substrate estrone sulfate. Regions involved in gain of CCK-8 transport by OATP1B1, when mapped to the crystal structures of bacterial transporters from the major facilitator superfamily, are positioned to suggest these regions could readily interact with drug substrates. Accordingly, our data provide new insight into the molecular determinants of the substrate specificity of these hepatic uptake transporters with relevance to targeted drug design and prediction of drug-drug interactions.

Contribution of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to hepatic uptake of nateglinide, and the prediction of drug–drug interactions via these transporters

We have investigated the contributions of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to the hepatic uptake of nateglinide, and the possibility of drug-drug interactions via these transporters. Uptake studies using transporter-expressing HEK293 cells and cryopreserved human hepatocytes were performed to examine the contributions of each transporter. Inhibition studies using cryopreserved human hepatocytes were performed to examine the possibility of drug-drug interactions. The rate of saturable hepatic uptake of nateglinide using human hepatocytes was 47.6%. A certain increase in uptake was observed in the examination using transporter-expressing HEK293 cells, indicating contributions of OATP1B1 and OATP1B3 to hepatic nateglinide uptake. The 50% inhibitory concentration (IC50) values of nateglinide using cryopreserved human hepatocytes for uptake of estrone 3-sulfate (substrate of OATP1B1), and cholecystokinin octapeptide (substrate of OATP1B3) were 168 and 17.4 µmol/l, respectively. Moreover, ciclosporin inhibited saturable hepatic uptake of nateglinide with an IC50 value of 6.05 µmol/l. The calculated 1 + I(in,max,u) /IC50 values for inhibition of OATP1B1 and OATP1B3 by nateglinide, and the inhibition of saturable uptake of nateglinide by ciclosporin, were all close to 1, indicating a low clinical risk of drug-drug interaction with nateglinide taken up via OATP1B1 and OATP1B3. OATP1B1 and OATP1B3 may have contributed to the hepatic uptake of nateglinide, but the possibility of drug-drug interactions appeared to be low.

Screening of the FDA approved anticancer drug library to identify novel substrates of organic anion transporting polypeptide 1B3

Organic anion transporting polypeptide (OATP) 1B3 is a liver-specific transport protein normally expressed in the basolateral membrane of hepatocytes. It transports a wide range of endo- and xenobiotics including some anticancer drugs. Recently expression of OATP1B3 has been demonstrated in several types of cancers. Thus, OATP1B3 is a potential target for anticancer drug transport into cancer cells. The aim of our study is to identify novel anticancer drugs that are substrates of OATP1B3. We used Chinese Hamster Ovary cells stably expressing OATP1B3 and measured uptake of the model substrates estradiol-17β-glucuronide and estrone-3-sulfate in the presence and absence of the 88 FDA approved anticancer drugs. For strong inhibitors, we determined inhibition kinetics. The strongest inhibitors were rapamycin, vinblastine, celecoxib, actinomycin D, teniposide, megestrol, vinorelbine and docetaxel. So far we identified rapamycin and actinomycin D as competitive inhibitors of OATP1B3-mediated uptake. Because competitive inhibitors are potential substrates of a given transporter we will test these inhibitors for direct uptake. In conclusion, OATP1B3 might be used as a potential target to deliver anticancer drugs into OATP1B3-expressing cancers. Supported by NIH grants RR021940, GM077336.

Interactions of Green Tea Catechins with Organic Anion-Transporting Polypeptides

Organic anion-transporting polypeptides (OATPs) are multispecific transporters that mediate the uptake of numerous drugs and xenobiotics into cells. Here, we examined the effect of green tea (Camellia sinensis) catechins on the function of the four OATPs expressed in human enterocytes and hepatocytes. Uptake of the model substrate estrone-3-sulfate by cells expressing OATP1A2, OATP1B1, OATP1B3, or OATP2B1 was measured in the absence and presence of the four most abundant flavonols found in green tea. Uptake by OATP1A2, OATP1B1, and OATP2B1 was inhibited by epicatechin gallate (ECG) and epigallocatechin gallate (EGCG) in a concentration-dependent way. In contrast, OATP1B3-mediated uptake of estrone-3-sulfate was strongly stimulated by EGCG at low substrate concentrations. The effect of EGCG on OATP1B3 was also studied with additional substrates: uptake of estradiol-17β-glucuronide was unchanged, whereas uptake of Fluo-3 was noncompetitively inhibited. Both ECG and EGCG were found to be substrates of OATP1A2 (K(m) values of 10.4 and 18.8 μM, respectively) and OATP1B3 (34.1 and 13.2 μM, respectively) but not of OATP1B1 or OATP2B1. These results indicate that two of the major flavonols found in green tea have a substantial effect on the function of OATPs expressed in enterocytes and hepatocytes and can potentially alter the pharmacokinetics of drugs and other OATP substrates. In addition, the diverse effects of EGCG on the transport of other OATP1B3 substrates suggest that different transport/binding sites are involved.

Transcellular movement of hydroxyurea is mediated by specific solute carrier transporters

Hydroxyurea has proven laboratory and clinical therapeutic benefits for sickle cell anemia and other diseases, yet many questions remain about its in vivo pharmacokinetic and pharmacodynamic profiles. Previous reports suggest that hydroxyurea passively diffuses across cells, but its observed rapid absorption and distribution are more consistent with facilitated or active transport. We investigated the potential role of solute carrier (SLC) transporters in cellular uptake and accumulation of hydroxyurea. Passive diffusion of hydroxyurea across cell membranes was determined using the parallel artificial membrane permeability assay. SLC transporter screens were conducted using in vitro intracellular drug accumulation and transcellular transport assays in cell lines and oocytes overexpressing SLC transporters. Gene expression of SLC transporters was measured by real-time polymerase chain reaction in human tissues and cell lines. Hydroxyurea had minimal diffusion across a lipid bilayer but was a substrate for five different SLC transporters belonging to the organic cation/carnitine transporters and organic anion transporting polypeptides (OATP) families of transporters and urea transporters A and B. Further characterization of hydroxyurea transport revealed that cellular uptake by OATP1B3 is time- and temperature-dependent and inhibited by known substrates of OATP1B3. Urea transporters A and B are expressed differentially in human tissues and erythroid cells, and transport hydroxyurea bidirectionally via facilitated diffusion. These studies provide new insight into drug transport proteins that may be involved in the in vivo absorption, cellular distribution, and elimination of hydroxyurea. Elucidation of hydroxyurea transcellular movement should improve our understanding of its pharmacokinetics and pharmacodynamics, and may help explain some of the interpatient drug variability observed in patients with sickle cell anemia.

High Impact of Oatp1a/1b Transporters on In Vivo Disposition of the Hydrophobic Anticancer Drug Paclitaxel

Organic anion-transporting polypeptides (OATP) mediate the cellular uptake of a broad range of drugs. The hydrophobic anticancer drug, paclitaxel (PTX), was recently identified as a substrate for OATP1B3 in vitro. We investigated the role of Oatp1a/1b transporters in the pharmacokinetics of PTX in vivo, as well as their impact at different dose levels of PTX and methotrexate (MTX). Recently generated Slco1a/1b(-/-) (lacking all Oatp1a/1b transporters) and wild-type mice were intravenously dosed with 2, 10, or 50 mg/kg of PTX, or with 10, 50, or 500 mg/kg of MTX, and plasma and tissue drug concentrations were measured. In spite of its hydrophobicity, PTX systemic exposure (at 10 mg/kg) was increased by greater than 2-fold in Slco1a/1b(-/-) mice compared with wild-type, whereas PTX liver uptake was reduced by about 2-fold. Oatp1a/1b transporters displayed a high impact on PTX and MTX pharmacokinetics over a broad dose range. For MTX, even at 500 mg/kg, saturation of Oatp1a/1b was not observed, with a 3.4-fold increase in plasma and 30-fold decrease in liver levels in Slco1a/1b(-/-) mice compared with wild-type. Although beginning saturation of Oatp1a/1b was observed at the highest dose of PTX, plasma levels in Slco1a/1b(-/-) mice were still 1.7-fold increased and liver levels 1.5-fold decreased compared with wild-type. Oatp1a/1b transporters play a pronounced role in determining plasma levels and tissue distribution of MTX and PTX, thus affecting even highly hydrophobic drugs. Variation in OATP1A/1B transporter activity, due to genetic variation, inhibition, and/or tumor expression might affect toxicity and therapeutic efficacy of these anticancer drugs.

Movement of Hydroxyurea Across Cell Membranes Is Mediated by Specific Solute Carrier (SLC) Transporters.

Abstract 2575Poster Board II-552 Background:Hydroxyurea is the only FDA-approved drug for the treatment of sickle cell anemia (SCA) in adults. Hydroxyurea increases fetal hemoglobin, decreases hospitalizations and painful events, and reduces mortality. With an oral bioavailability of > 90%, hydroxyurea is rapidly absorbed and distributed throughout the body. Though hydroxyurea has proven to be effective in treating SCA, there is considerable inter-patient variability observed in the pharmacokinetics and pharmacodynamics of hydroxyurea. Currently, mechanisms involved in the absorption, distribution, and elimination of hydroxyurea remain unclear. Recently, key transmembrane proteins have been identified as drug transporters due to their ability to move a variety of xenobiotic substances across cell membranes. Drug transporters are widely distributed throughout the body, and most are specific to certain substrates. Solute carrier (SLC) transporters in particular have been to shown to significantly impact drug pharmacokinetics by influencing the absorption, distribution, and elimination of specific drugs. The present study was designed to identify SLC transporters that may influence the absorption, distribution, and/or elimination of hydroxyurea in patients with SCA. Methods:In vitro studies using an equilibrium dialysis plate were performed to determine the amount of hydroxyurea that binds to human serum proteins. Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [14C]-hydroxyurea accumulation in HEK293 cells and oocytes that overexpress organic anion transporters (OAT1-3), organic cation transporters (OCT1-3), organic cation/carnitine transporters (OCTN1-2), organic anion transporting polypeptides (OATP1A2/OATP1B1/OATP1B3), or vector control. LLC-PK1 cells that overexpress urea transporters A or B (UTA/UTB) were used to determine UTA/UTB mediated transcellular transport of hydroxyurea in transwell plates. The transport of [14C]-hydroxyurea from apical to basal or from basal to apical compartments was measured for the UTA/UTB overexpressing cells and compared to vector control. UTA and UTB mRNA expression was measured by real-time PCR of cDNA obtained from human tissue samples. Results:Protein binding assays showed that >76% of [14C]-hydroxyurea remained unbound to proteins in human serum containing hydroxyurea at concentrations ranging from 1.5μM to 500μM. The fraction of unbound hydroxyurea was similar using serum obtained from pediatric patients with SCA. In uptake studies, [14C]-hydroxyurea was a potent substrate for OATP1B3 with an approximately 2-fold increase in drug accumulation compared to control (p<0.001). In contrast, hydroxyurea was found to be a weak substrate for OCTN1, OCTN2, OATP1A2, and OATP1B1 with only a 1.3-fold increase in drug accumulation compared to control (p<0.04). Transcellular transport of hydroxyurea was increased 3- and 2-fold by UTA and UTB, respectively, compared to vector control demonstrating hydroxyurea to be a potent substrate for these transporters as well (p<0.02). When the urea transporter inhibitor dimethylurea was added, hydroxyurea transport by UTA and UTB-expressing cells was decreased to levels observed with the vector control. In real-time PCR assays, kidney, muscle, and small intestine were among human tissues with high expression of UTA mRNA, while prostate, brain, and bone marrow had high levels of UTB mRNA expression. Conclusion:Cellular uptake of hydroxyurea is mediated by active transport via specific SLC transporters OATP1B3, UTA and UTB, which are expressed in liver, kidney, brain, intestine, and blood cells. Studies to further characterize hydroxyurea transporters should improve our understanding of the pharmacokinetic and pharmacodynamic profiles of hydroxyurea used in clinical practice for patients with SCA. Disclosures:No relevant conflicts of interest to declare.

Effect of mutations at Y358 and S548 on OATP1B3 mediated transport

Organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are liver specific transporters that share 80 % amino acid identity. However, only OATP1B3 transports cholecystokinin‐8 (CCK‐8). Previous docking studies with an OATP1B3 model suggested that Y358 and S548 could form hydrogen bonds with CCK‐8. Therefore, we tested the hypothesis that Y358 and S548 are important for OATP1B3 mediated CCK‐8 transport by replacing these amino acids. Site‐directed mutagenesis was performed to construct the Y358F and the S548A mutants. The cDNAs were transiently expressed in HEK293 cells and surface expression and function was determined. Both mutants were synthesized and their surface expression was slightly reduced as compared to OATP1B3. Transport of CCK‐8 by Y358F was reduced to 70 % of wild‐type OATP1B3 while S548A did not affect CCK‐8 transport. In order to investigate whether this effect was specific for CCK‐8 or a general phenomenon for OATP1B3 mediated transport we measured uptake of the additional OATP1B3 substrates bromosulfophthalein, estradiol‐17β‐glucuronide and taurocholate. Uptake of all substrates was normal for S548A. Mutant Y358F had normal bromosulfophthalein transport but uptake of both estradiol‐17β‐glucuronide and taurocholate was reduced to 40 %. Mutation of Y358 seems to affect OATP1B3 in a substrate dependent way but kinetic studies are required to definitively characterize these effects.

Molecular determinants for substrate selectivity of OATP1B3

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are liver‐specific transporters located at the basolateral membrane of hepatocytes and mediate uptake of amphipathic organic compounds from blood into hepatocytes. The amino acid sequence of OATP1B3 is 80% identical to that of OATP1B1 and the two transporters have common as well as distinct substrates. At low concentrations, estrone‐3‐sulfate is preferentially transported by OATP1B1, while cholecystokinin octapeptide (CCK‐8) is a specific substrate for OATP1B3. To determine which domains are important for the substrate selectivity of OATP1B3 we constructed a series of chimeric proteins between OATP1B1 and 1B3. These chimeras were transiently expressed in human embryonic kidney cells (HEK293), uptake of estrone‐3‐sulfate and CCK‐8 was measured and surface expression was determined. Most of the chimeras could be expressed and detected at the cell surface. A first set of chimeras revealed that the substrate recognition/transport site is within the second half of the protein. Using additional chimeras, we found that when OATP1B3 contained transmembrane domain 10 and extracellular loop 5 of OATP1B1 it was not able to efficiently transport CCK‐8. Thus, we conclude that the region around transmembrane domain 10 is crucial for recognition and/or transport of the OATP1B3‐specific substrate CCK‐8.

Ethics guidelines for clinical trials to be revised

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: <i>SLCO1A2</i>, <i>SLCO2B1</i>, <i>SLCO1B1</i>, <i>SLCO3A1</i>, <i>SLCO4A1</i>, <i>SLCO1B3</i>, <i>SLC22A7</i>, <i>SLC22A8</i>, <i>SLC22A1, SLC10A1</i>, <i>SLC15A1</i>, and <i>SLC15A2.</i> B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of <i>SLCO1B1</i>, <i>SLCO3A1</i>, and <i>SLCO1B3</i> was greatly repressed in HepG2 cells, whereas <i>SLCO2B1</i>, <i>SLC22A7</i>, and <i>SLC22A8</i> expression was either maintained or increased. On the contrary, in Chang liver cells, <i>SLC22A7</i> and <i>SLC22A8</i> genes were undetectable, whereas the expression of <i>SLCO3A1</i>, <i>SLCO4A1</i>, and <i>SLCO1B3</i> was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Establishment of a Set of Double Transfectants Coexpressing Organic Anion Transporting Polypeptide 1B3 and Hepatic Efflux Transporters for the Characterization of the Hepatobiliary Transport of Telmisartan Acylglucuronide

In the hepatic uptake of organic anions, organic anion transporting polypeptide (OATP) 1B1 is believed to be mainly involved. We have constructed a set of double-transfected cells coexpressing OATP1B1 and hepatic efflux transporters and characterized the transcellular transport of several anions. Recent reports have also suggested the importance of OATP1B3 in the hepatic uptake of some compounds. However, there is little information about OATP1B3-selective substrate and no good tool for the evaluation of efflux transporters of OATP1B3 substrates. In the present study, we found an OATP1B3-selective substrate and established a novel set of double transfectants expressing OATP1B3. Telmisartan acylglucuronide (tel-glu) is a main metabolite of telmisartan, an angiotensin II receptor antagonist. Tel-glu is recognized by hepatobiliary transport systems and efficiently distributed to liver. Several studies using rat and human hepatocytes and transporter-expressing cells revealed that OATP1B3 was responsible for the hepatic uptake of tel-glu in humans. By using double transfectants expressing OATP1B3, we investigated the transcellular transport of tel-glu as well as estradiol 17beta-d-glucuronide (E(2)17betaG) and cholecystokinin octapeptide (CCK-8) to identify the responsible efflux transporters in their biliary excretion. Vectorial basal-to-apical transport of tel-glu was observed in all kinds of double transfectants expressing OATP1B3. In contrast, basal-to-apical transport of E(2)17betaG and CCK-8 was seen only in the OATP1B3/MRP2 double transfectant compared with OATP1B3-expressing cells. Therefore, the newly established set of double transfectants expressing OATP1B3 combined with OATP1B1-expressing double transfectants can be used as a powerful tool for the rapid identification of hepatic uptake and efflux transporters of organic anions.

Influence of SLCO1B3 Gene Polymorphism on the Pharmacokinetics of Digoxin in Terminal Renal Failure

Digoxin (DX) is mainly excreted unchanged in the urine. In patients undergoing hemodialysis (HD patients), the relative contribution of hepatic elimination is increased. DX is a substrate of OATP1B3 (SLCO1B3), expressed on the sinusoidal membranes of hepatocytes in humans. Therefore, we investigated the relationship between SLCO1B3 gene polymorphisms and the value of trough concentration-to-dose ratio (C/D ratio) of DX in HD patients. We investigated two deletion polymorphisms in complete linkage disequilibrium (-28 to -11 and -7 to -4) and two SNPs in complete linkage disequilibrium (T334G and G699A). Blood was sampled 62-72 hours after the oral administration of DX. The C/D ratio of DX was lower in patients with the deletion allele than in those homozygous for the insertion allele, and was lower in patients with the 334T/669G allele than in those homozygous for the 334G/699A allele, although the differences were not statistically significant. The C/D ratio of DX was significantly higher in patients with homozygous for the insert-variant allele (median: 121.8 (ng/mL)/(mg/week/kg), range: 92.5-259.4 (ng/mL)/(mg/week/kg) than in others (median: 93.4 (ng/mL)/(mg/week/kg), range: 66.5-154.3 (ng/mL)/(mg/week/kg)). In conclusion, the insert-variant allele of the OATP1B3 gene may increase the C/D ratio of DX in HD patients.

Rapid screening of antineoplastic candidates for the human organic anion transporter OATP1B3 substrates using fluorescent probes

A rapid screening system has been established to extract novel candidates that exhibit potent inhibition of the transport of fluorescent substrate by organic anion transporting polypeptide (OATP) 1B3. OATP1B3 is abundantly expressed in solid digestive organ cancers. Thus, the identification of new substrates leads to novel strategies for effective cancer chemotherapy with minimal adverse effects. We used an automated image acquisition and analysis system (IN Cell Analyzer 1000) to visualize the transport and subsequent accumulation of the fluorescent substrate chenodeoxycholyl-(Nε-NBD)-lysine (CDCA-NBD). Antineoplastic screening demonstrated that five candidates agents, docetaxel, actinomycin D, mitoxantrone, paclitaxel, and SN-38, exhibited potent inhibitory effects on OATP1B3-mediated transport of CDCA-NBD. To clarify if these antineoplastic drugs are substrates for OATP1B3, we performed transport assays in OATP1B3-expressing cells. We determined that SN-38 is a novel substrate for OATP1B3. In conclusion, our results demonstrate that the screening system established in this study is a useful method for the rapid extraction of candidate therapeutic agents from the large numbers of compounds.

Molecular Determinants in the Transport of a Bile Acid-Derived Diagnostic Agent in Tumoral and Nontumoral Cell Lines of Human Liver

Contrast-enhanced magnetic resonance imaging (CE-MRI) is a valuable technique for the diagnosis of liver diseases. As gadocoletic acid trisodium salt (B22956/1), a new contrast agent showing high biliary excretion, may be potentially advantageous in hepatobiliary imaging, the aim of the study was to investigate the molecular mechanisms of hepatic transport of the B22956 ion in a cellular model of hepatic tumor. B22956 ion uptake was measured in tumoral (HepG2) and nontumoral (Chang liver) hepatic cell lines. Absolute quantitative real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) analyses, using cloned PCR products as standards, were performed on total RNA of both cell lines and normal liver to evaluate the transcription of 12 transport genes: SLCO1A2, SLCO2B1, SLCO1B1, SLCO3A1, SLCO4A1, SLCO1B3, SLC22A7, SLC22A8, SLC22A1, SLC10A1, SLC15A1, and SLC15A2. B22956 transport was more efficient in Chang liver than in HepG2 cells and was inhibited by cholecystokinin-8, a specific substrate of OATP1B3. Real-time RT-PCR analyses revealed different transcription profiles in the tumoral and nontumoral cell lines. Compared with normal liver, the expression of SLCO1B1, SLCO3A1, and SLCO1B3 was greatly repressed in HepG2 cells, whereas SLCO2B1, SLC22A7, and SLC22A8 expression was either maintained or increased. On the contrary, in Chang liver cells, SLC22A7 and SLC22A8 genes were undetectable, whereas the expression of SLCO3A1, SLCO4A1, and SLCO1B3 was similar to normal liver. Transport studies and gene expression analyses indicated that B22956 ion is a good substrate to the liver-specific OATP1B3, reported to be poorly expressed or absent in human liver tumors. Therefore, B22956 may be helpful in detecting hepatic neoplastic lesions by CE-MRI.

Molecular Characterization and Inhibition of Amanitin Uptake into Human Hepatocytes

Amatoxins are the main poison of the green death cap (Amanita phalloides) and among the most dangerous natural toxins causing hepatic failure. A possible therapeutic approach is the inhibition of the transporting systems mediating the uptake of amatoxins into human hepatocytes, which, however, have yet to be identified. In the current study we tested whether members of the organic anion-transporting polypeptide (OATP) family, localized in the sinusoidal membranes of human hepatocytes, are involved in amatoxin uptake. For this, Madin Darby canine kidney strain II (MDCKII) cells stably expressing human OATP1B3, OATP2B1, or OATP1B1, were assayed for the uptake of 3H-labeled O-methyl-dehydroxymethyl-alpha-amanitin. Under our conditions, only OATP1B3 was able to transport amanitin with a K(m) value of 3.7 microM +/- 0.6 microM. Accordingly, toxin uptake was inhibited by OATP1B3 substrates and inhibitors (cyclosporin A, rifampicin, the quinoline derivatives MK571 ([(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl)((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid]) and montelukast, the cholecystokinin octapeptide (CCK-8), paclitaxel, and bromosulfophthalein), as well as by some antidotes used in the past for the treatment of human amatoxin poisoning (silibinin dihemisuccinate, penicillin G, prednisolone phosphate, and antamanide). These transport studies are in line with viability assays monitoring the toxic effect of amanitin on the transfected MDCKII cells. Further support for amatoxin transport was found in primary human hepatocytes, expressing OATP1B3, OATP2B1, and OATP1B1, where CCK-8, a substrate specific for OATP1B3, prevented the fragmentation of nucleoli, a lesion typical for amanitin action. In conclusion, we have identified OATP1B3 as the human hepatic uptake transporter for amatoxins; moreover, substrates and inhibitors of OATP1B3, among others rifampicin, may be useful for the treatment of human amatoxin poisoning.

Human Hepatobiliary Transport of Organic Anions Analyzed by Quadruple-Transfected Cells

Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identification of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes.