Human OATP2B1 Research Articles

Impact of OATP2B1 on Pharmacokinetics of Atorvastatin Investigated in rSlco2b1-Knockout and SLCO2B1-Knockin Rats.

The organic anion transporting polypeptide (OATP) 2B1 is considered an emerging drug transporter that is found expressed in pharmacokinetically relevant organs such as the liver, small intestine, and kidney. Despite its interaction with various substrate drugs, the understanding of its in vivo relevance is still limited. In this study, we first validated the interaction of atorvastatin with rat OATP2B1 using transiently transfected HeLa cells. Moreover, we characterized our rSlco2b1-knockout and SLCO2B1-knockin rats for mRNA, protein expression, and localization of OATP2B1 in the liver, small intestine, and kidney. The transporter showed the highest expression in the liver followed by the small intestine. In humanized rats, human OATP2B1 is localized on the sinusoidal membrane of hepatocytes. In enterocytes of wild-type and humanized rats, the transporter was detected in the luminal membrane with the vast majority being localized subapical. Subsequently, we assessed atorvastatin pharmacokinetics in male wild-type, rSlco2b1-knockout, and SLCO2B1-knockin rats after a single-dose administration (orally and intravenously). Investigating the contribution of rat OATP2B1 or human OATP2B1 to oral atorvastatin pharmacokinetics revealed no differences in concentration-time profiles or pharmacokinetic parameters. However, when comparing the pharmacokinetics of atorvastatin after intravenous administration in SLCO2B1-humanized rats and knockout animals, notable differences were observed. In particular, the systemic exposure (area under the curve) decreased by approximately 40% in humanized animals, whereas the clearance was 57% higher in animals expressing human OATP2B1. These findings indicate that human OATP2B1 influences pharmacokinetics of atorvastatin after intravenous administration, most likely by contributing to the hepatic uptake. SIGNIFICANCE STATEMENT: Wild-type, rSlco2b1-knockout, and SLCO2B1-humanized Wistar rats were characterized for the expression of rat and human SLCO2B1/OATP2B1. Pharmacokinetic studies of atorvastatin over 24 hours were conducted in male wild-type, rSlco2b1-knockout, and SLCO2B1-humanized rats. After a single-dose intravenous administration, a lower systemic exposure and an increase in clearance were observed in SLCO2B1-humanized rats compared with knockout animals indicating a contribution of OATP2B1 to the hepatic clearance.

Organic anion-transporting polypeptide 2B1 knockout and humanized mice; insights into the handling of bilirubin and drugs

Organic anion transporting polypeptide 2B1 (OATP2B1/SLCO2B1) facilitates uptake transport of structurally diverse endogenous and exogenous compounds. To investigate the roles of OATP2B1 in physiology and pharmacology, we established and characterized Oatp2b1 knockout (single Slco2b1-/- and combination Slco1a/1b/2b1-/-) and humanized hepatic and intestinal OATP2B1 transgenic mouse models. While viable and fertile, these strains exhibited a modestly increased body weight. In males, unconjugated bilirubin levels were markedly reduced in Slco2b1-/- compared to wild-type mice, whereas bilirubin monoglucuronide levels were modestly increased in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice. Single Slco2b1-/- mice showed no significant changes in oral pharmacokinetics of several tested drugs. However, markedly higher or lower plasma exposure of pravastatin and the erlotinib metabolite OSI-420, respectively, were found in Slco1a/1b/2b1-/- compared to Slco1a/1b-/- mice, while oral rosuvastatin and fluvastatin behaved similarly between the strains. In males, humanized OATP2B1 strains showed lower conjugated and unconjugated bilirubin levels than control Slco1a/1b/2b1-deficient mice. Moreover, hepatic expression of human OATP2B1 partially or completely rescued the impaired hepatic uptake of OSI-420, rosuvastatin, pravastatin, and fluvastatin in Slco1a/1b/2b1-/- mice, establishing an important role in hepatic uptake. Expression of human OATP2B1 in the intestine was basolateral and markedly reduced the oral availability of rosuvastatin and pravastatin, but not of OSI-420 and fluvastatin. Neither lack of Oatp2b1, nor overexpression of human OATP2B1 had any effect on fexofenadine oral pharmacokinetics. While these mouse models still have limitations for human translation, with additional work we expect they will provide powerful tools to further understand the physiological and pharmacological roles of OATP2B1.

Investigating the interactions of flavonoids with human OATP2B1: inhibition assay, IC50 determination, and structure-activity relationship analysis.

Human organic anion transporting polypeptide 2B1 (OATP2B1) is a crucial transporter for the absorption and disposition of many drugs. Its inhibition by small molecules may alter the pharmacokinetic profile of its substrate drugs. In this study, the interactions of 29 common flavonoids with OATP2B1 were explored using the fluorescent substrate 4',5'-dibromofluorescein and structure-activity relationship analysis. Our results showed that flavonoid aglycones interact with OATP2B1 more strongly than their 3-O- and 7-O-glycoside counterparts, as hydrophilic and bulky groups at these two sites are detrimental to flavonoids' binding with OATP2B1. In contrast, hydrogen-bond forming groups at the C-6 position of ring A and the C-3' and C-4' positions of ring B could strengthen the interaction of flavonoids with OATP2B1. However, a hydroxyl or sugar moiety at the C-8 position of ring A is unfavorable. Our results also indicated that flavones usually interact more strongly with OATP2B1 than their 3-hydroxyflavones (flavonols). The obtained information could be useful for the prediction of additional flavonoids for their interaction with OATP2B1.

Zebrafish (Danio rerio) Oatp2b1 as a functional ortholog of the human OATP2B1 transporter.

OATP2B1 belongs to a highly conserved organic anion transporting polypeptide (OATP) family of transporters, involved in the cellular uptake of both endogenous and exogenous compounds. The reported substrates of human OATP2B1 include steroid conjugates, bile salts, and thyroid hormones, as well as pharmaceuticals. Human OATP2B1 has orthologous genes in other vertebrate species, including zebrafish (Danio rerio), a widely used model organism in biomedical and environmental research. Our previous studies showed that zebrafish Oatp2b1 was phylogenetically closest to mammalian OATP2B1/Oatp2b1 and that it shares a similar tissue expression pattern. In this study, we aimed at discovering whether zebrafish Oatp2b1 could be a functional ortholog of human and rodent OATP2B1. To test this hypothesis, our primary goal was to obtain the first in vitro and in silico insights related to the structure and potential substrate preferences of zebrafish Oatp2b1. We generated cells transiently and stably transfected with zebrafish Oatp2b1 cloned from zebrafish liver, constructed an Oatp2b1 homology model, developed transport activity assays with model fluorescent substrate Lucifer yellow, and finally utilized this assay to analyze the interaction of zebrafish Oatp2b1 with both physiological and xenobiotic substances. Apart from structure similarities, our data revealed the strongest interaction of zebrafish Oatp2b1 with bile acids, steroid sulfates, thyroid hormones, and bilirubin, as well as xenobiotics bromosulfophthalein and sulfasalazine, which indicates its functional orthology with human OATP2B1.

Post-transcriptional regulation of OATP2B1 transporter by a microRNA, miR-24

Human OATP2B1, a member of organic anion transporting polypeptide family, is expressed in several tissues, including small intestine and liver, and contributes to cellular uptake of endogenous compounds and various drugs. Altered expression of OATP2B1 affects pharmacokinetics of substrate drugs; however, limited information is available on the regulation of OATP2B1 expression. This study aimed to explore microRNAs (miRNAs) that regulate OATP2B1 expression using HEK293 cells transfected with an expression plasmid of OATP2B1 including 3′-UTR (HEK/OATP2B1) and Caco-2 as a model of human intestine. Computational analysis predicted that three miRNAs, miR-143, miR-125b and miR-24, may bind to the 3′-UTR of OATP2B1 mRNA. A luciferase assay using a plasmid containing the 3′-UTR of OATP2B1 gene demonstrated that only miR-24 significantly reduced its expression. The overexpression of miR-24 decreased the expression of OATP2B1 mRNA and protein in HEK/OATP2B1 and Caco-2 cells and uptake of [3H]estrone-3-sulfate by HEK/OATP2B1 cells. However, a statistically significant increase of endogenous OATP2B1 expression was not observed by miR-24 inhibitor in Caco-2 cells. In conclusion, it was found that miR-24 negatively regulates OATP2B1 expression, resulting in suppression of OATP2B1 activity, while its contribution to regulation of apparent expression of OATP2B1 is considered to depend on tissues and cell types.

Structural dissection of 13-epiestrones based on the interaction with human Organic anion-transporting polypeptide, OATP2B1

Human OATP2B1 encoded by the SLCO2B1 gene is a multispecific transporter mediating the cellular uptake of large, organic molecules, including hormones, prostaglandins and bile acids. OATP2B1 is ubiquitously expressed in the human body, with highest expression levels in pharmacologically relevant barriers, like enterocytes, hepatocytes and endothelial cells of the blood-brain-barrier. In addition to its endogenous substrates, OATP2B1 also recognizes clinically applied drugs, such as statins, antivirals, antihistamines and chemotherapeutic agents and influences their pharmacokinetics. On the other hand, OATP2B1 is also overexpressed in various tumors. Considering that elevated hormone uptake by OATP2B1 results in increased cell proliferation of hormone dependent tumors (e.g. breast or prostate), inhibition of OATP2B1 can be a good strategy to inhibit the growth of these tumors.13-epiestrones represent a potential novel strategy in the treatment of hormone dependent cancers by the suppression of local estrogen production due to the inhibition of the key enzyme of estrone metabolism, 17ß-hydroxysteroid-dehydrogenase type 1 (HSD17ß1). Recently, we have demonstrated that various phosphonated 13-epiestrones are dual inhibitors also suppressing OATP2B1 function. In order to gain better insights into the molecular determinants of OATP2B1 13-epiestrone interaction we investigated the effect of C-2 and C-4 halogen or phenylalkynyl modified epiestrones on OATP2B1 transport function. Potent inhibitors (with EC50 values in the low micromolar range) as well as non-inhibitors of OATP2B1 function were identified. Based on the structure-activity relationship (SAR) of the various 13-epiestrone derivatives we could define structural elements important for OATP2B1 inhibition. Our results may help to understand the drug/inhibitor interaction profile of OATP2B1, and also may be a useful strategy to block steroid hormone entry into tumors.

Clinically Relevant OATP2B1 Inhibitors in Marketed Drug Space.

OATP2B1 is an intestinal and hepatic drug uptake transporter. Intestinal OATP2B1 has been elucidated as the mechanism of unexpected clinical drug-drug interactions (DDIs), where drug exposure was unexpectedly decreased with unchanged half-life. Hepatic OATP2B1 may be an understudied clinical DDI mechanism. The aim of the present work was to understand the prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors in marketed drug space. HEK293 cells stably overexpressing human OATP2B1 or vector control were generated and cultured for 72 h in a 96-well format. OATP2B1-mediated uptake of dibromofluorescein (DBF) was found to be optimal at 10 μM concentration and 30 min incubation time. A total of 294 drugs (top 300 marketed drugs, excluding biologics and restricted drugs, supplemented with ∼100 small-molecule drugs) were screened for OATP2B1 inhibition at 10 μM. Drugs demonstrating ≥50% inhibition in this screen were advanced for IC50 determination, which was extrapolated to clinical intestinal and hepatic OATP2B1 inhibition as per 2017 FDA DDI guidance. Of the 294 drugs screened, 67 elicited ≥50% inhibition of OATP2B1-mediated DBF uptake at 10 μM screening concentration. For the 67 drugs flagged in the single-concentration inhibition screen, upon evaluation of a full concentration range, IC50 values could be determined for 58 drugs. OATP2B1 IC50 values established for these 58 drugs were extrapolated as potentially clinically relevant at the intestinal level for 38 orally administered drugs (Igut/IC50 ≥ 10), and 17 were flagged as potential clinical inhibitors of hepatic OATP2B1 uptake (1 + Iin,max,u/IC50 ≥ 1.1). This analysis of 294 drugs demonstrated prevalence of clinically relevant intestinal and hepatic OATP2B1 inhibitors to be 13 and 6%, respectively. As OATP2B1-inhibitor drugs are not exceedingly rare, these results suggest that clinical OATP2B1 DDIs have been rarely observed because OATP2B1 is uncommonly the predominant determinant of drug disposition.

Role of OATP2B1 in Drug Absorption and Drug‐Drug Interactions

BackgroundThe human organic anion‐transporting polypeptide 2B1 (OATP2B1; SLC21A9) is localized to the basolateral membrane of hepatocytes and the brush‐border membrane of small‐intestinal enterocytes. Based on its distribution pattern and functional similarity to OATP1B‐type transporters, it has been hypothesized that OATP2B1 might influence the absorption and disposition properties of a wide range of xenobiotic agents. Although several prescription drugs, including statins such as fluvastatin, have been identified as OATP2B1 substrates in vitro, the lack of a useful in vivo model system has limited our understanding of the pharmacological role of this transporter.Experimental designOATP2B1‐overexpressing cells were generated by transfecting HEK293 cells with an empty vector (VC) or a construct containing human OATP2B1 or mouse OATP2B1 cDNA. Transporter function was evaluated in vitro by measuring the comparative cellular uptake of [3H]estrone‐3‐sulfate (ES) in VC cells and OATP2B1‐overexpressing cells. Candidate substrates and inhibitors were identified based on the difference in intracellular accumulation of radioactivity. OATP2B1‐knockout (KO) mice were obtained from the Slco2b1tm1a(KOMP)Wtsi clone, and derived chimera and subsequent progeny were mated to C57BL/6N for germline production and colony expansion. Mouse genotypes were confirmed by PCR, and OATP2B1 gene deletion was verified by qPCR on major organs. The pharmacokinetic profiles of fluvastatin, a known OATP2B1 substrate, and sorafenib, a known OATP2B1 inhibitor, were determined through serial blood collection and subsequent LC‐MS/MS analysis. Noncompartmental pharmacokinetic parameters were calculated using WinNolin 6.2 software (Pharsight).ResultsAn approximately four‐fold increase in intracellular accumulation of radiolabeled ES (2.5 μM) was observed in the OATP2B1 overexpressing cells compared to VC cells (34.8 ± 2.45 pmol/mg vs 8.38 ± 0.341 pmol/mg, respectively). Subsequently, we identified several drugs as candidate OATP2B1 substrates, including multiple tyrosine kinase inhibitors (TKIs). Knockout of OATP2B1 in organs from KO mice was confirmed at the gene level (P<0.01), and consistent with the intestinal localization of OATP2B1, we found that the absorption of fluvastatin was affected by OATP2B1 deficiency (Tmax: 1 h vs 0.5 h; Cmax: 1.05 μg/mL vs 2.19 μg/mL). No gender differences in mRNA expression levels were detected. Among 24 different agents tested, sorafenib was identified as one of the most potent OATP2B1 inhibitors in the class of TKIs. However, the pharmacokinetic properties of sorafenib and its main metabolites were not significantly affected by OATP2B1 deficiency.ConclusionOur studies have identified novel potent inhibitors of OATP2B1 among TKIs, a rapidly expanding class of drugs used in various therapeutic areas. The availability of a viable, well‐characterized OATP2B1‐deficient mouse model provides a previously unavailable opportunity to unequivocally determine the contribution of this transporter to the absorption and drug‐drug interaction potential of drugs.Support or Funding InformationEli Lilly Fellowship in PharmaceuticsThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction of soy isoflavones and their main metabolites with hOATP2B1 transporter.

Membrane organic anion-transporting polypeptides (OATPs) are responsible for the drug transmembrane transport within the human body. The function of OATP2B1 transporter can be inhibited by various natural compounds. Despite increased research interest in soya as a part of human diet, the effect of its active components to interact with hOATP2B1 has not been elucidated in a complex extent. This in vitro study examined the inhibitory effect of main soy isoflavones (daidzin, daidzein, genistin, genistein, glycitin, glycitein, biochanin A, formononetin) and their metabolites formed in vivo (S-equol, O-desmethylangolensin) towards human OATP2B1 transporter. MDCKII cells overexpressing hOATP2B1 were employed to determine quantitative inhibitory parameters of the tested compounds and to analyze mechanism/s of the inhibitory interaction. The study showed that aglycones of soy isoflavones and the main biologically active metabolite S-equol were able to significantlyinhibit hOATP2B1-mediated transport. The Ki values for most of aglycones range from 1 to 20μM. In contrast, glucosides did not exhibit significant inhibitory effect. The kinetic analysis did not indicate a uniform type of inhibition towards the hOATP2B1 although predominant mechanism of inhibition seemed to be competitive. These findings may suggest that tested soy isoflavones and their metabolites might affect transport of xenobiotics including drugs across tissue barriers via hOATP2B1.

Hepatic Metabolism and Disposition of Baicalein via the Coupling of Conjugation Enzymes and Transporters—In Vitro and In Vivo Evidences

Baicalein (Ba) was found to be subject to serious first-pass metabolism after oral administration. We previously revealed the important role of intestine in the low oral bioavailability of Ba. The present study aims to evaluate the hepatic metabolism and disposition of Ba. Ba was given to Sprague-Dawley rats through bolus or infusion via intravenous or intra-portal route of administrations. Both plasma and bile samples at different time intervals were obtained. Concentrations of Ba and potential metabolites in the collected samples were analyzed with HPLC/UV and identified by LC/MS/MS, respectively. Plasma concentration versus time profiles of Ba obtained from intravenous and intra-portal administrations were compared to estimate the extent of hepatic metabolism. In addition, transport studies of baicalein-7-glucuronide (BG), one of the major metabolites of Ba, were carried out using transfected cell systems overexpressing various human organic anion-transporting polypeptide (OATP) isoforms to estimate the specific transporters involved in the hepatic disposition of Ba metabolites. The results showed that liver, in addition to intestine, also conferred extensive metabolism to Ba. Several mono- and di-conjugates of Ba, which were mainly glucuronides, sulfates, and methylates, were found in bile. The transport study demonstrated that besides MRPs and BCRP, human OATP2B1 and OATP1B3 in liver might also mediate the secretion of BG to bile. In summary, liver plays an important role in the metabolism of Ba and transport of its conjugated metabolites.

Species dependent substrate specificity of the human and mouse OATP2B1/Oatp2b1

Knockout mouse models are valuable tools to investigate the role of transporters in drug disposition and hepatotoxicity. However, due to limited understanding of species differences in substrate specificity, extrapolation of transporter data between species can be problematic. In vitro expression systems are a critical tool to screen for substrates and to delineate differences between human and mouse transporters. Therefore, the objective of the current study was to compare the substrate specificities of human OATP2B1 to mouse Oatp2b1. We used a CHO cell line stably expressing OATP2B1 and HEK293 cells transiently expressing Oatp2b1 and measured uptake of radiolabeled substrates. Our results confirm the model compounds estrone‐3‐sulfate and bromosulfophthalein as substrates for both the human and the mouse transporter. Additionally, we identified the anticancer drug paclitaxel and the anti‐HIV drug saquinavir as substrates for human OATP2B1. Mouse Oatp2b1 demonstrated a broader substrate specificity and transported numerous substrates including docetaxel, oleic acid, methotrexate, paclitaxel, ritonavir and saquinavir. Based on these data we conclude that the OATP2B sub‐family contains multispecific xenobiotic transporters with the mouse Oatp2b1 having a broader substrate specificity than its human ortholog.

Expression, purification and characterization of the human membrane transporter protein OATP2B1 from Sf9 insect cells

OATP2B1 is an important member of the organic anion transporting polypeptides (OATP) family and is implicated in the intestinal and hepatic disposition of endo- and xenobiotics. The purpose of this work was to produce a highly purified protein for use as a reference standard for quantification of OATP2B1 in human tissue and in vitro assay systems. Here, we report the successful expression, purification and characterization of OATP2B1 in a heterologous expression system. Protein expressed by the Sf9-baculovirus expression system is functionally active as demonstrated by saturable uptake kinetics with a Km of 5.9±0.76μM for estrone-3-sulfate. OATP2B1 was extracted from Sf9-membranes with ABS-14-4 detergent and purified using a one-step FLAG-tag purification method. Yield of OATP2B1 from Sf9 cells was 1.1mg per liter of culture, for a final recovery of 1.8%. SDS–PAGE resolution and Western blot of purified protein displayed multiple banding of OATP2B1-specific protein, which was thoroughly investigated to confirm homogeneity of the sample. C-terminal FLAG-tag purification and immunoblot detection, together with N-terminal sequencing, confirmed the presence of only full-length protein. Treatment with endoglycosidases had little effect on the migration pattern in SDS–PAGE, suggesting that multiple banding was not due to different glycosylation states of the protein. Amino acid analysis further confirmed the homogeneity of the protein with a calculated extinction coefficient of 80,387cm−1M−1. Physical, biochemical and functional characterization show that purified human OATP2B1 is pure, homogeneous and appropriate for use as a standard to quantitate expression of OATP2B1 in in vitro systems and tissue samples.

Role of Transporters in the Disposition of the Selective Phosphodiesterase-4 Inhibitor (+)-2-[4-({[2-(Benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbonyl]-amino}-methyl)-3-fluoro-phenoxy]-propionic Acid in Rat and Human

The role of transporters in the disposition of (+)-2-[4-({[2-(benzo[1,3]dioxol-5-yloxy)-pyridine-3-carbonyl]-amino}-methyl)-3-fluoro-phenoxy]-propionic acid (CP-671,305), an orally active inhibitor of phosphodiesterase-4, was examined. In bile duct-exteriorized rats, a 7.4-fold decrease in the half-life of CP-671,305 was observed, implicating enterohepatic recirculation. Statistically significant differences in CP-671,305 pharmacokinetics (clearance and area under the curve) were discernible in cyclosporin A- or rifampicin-pretreated rats. Considering that cyclosporin A and rifampicin inhibit multiple uptake/efflux transporters, the interactions of CP-671,305 with major human hepatic drug transporters, multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistant protein (BCRP), and organic anion-transporting polypeptide (OATPs) were evaluated in vitro. CP-671,305 was identified as a substrate of MRP2 and BCRP, but not MDR1. CP-671,305 was a substrate of human OATP2B1 with a high affinity (Km = 4 microM) but not a substrate for human OATP1B1 or OATP1B3. Consistent with these results, examination of hepatobiliary transport of CP-671,305 in hepatocytes indicated active uptake followed by efflux into bile canaliculi. Upon examination as a substrate for major rat hepatic Oatps, CP-671,305 displayed high affinity (Km = 12 microM) for Oatp1a4. The role of rat Mrp2 in the biliary excretion was also examined in Mrp2-deficient rats. The observations that CP-671,305 pharmacokinetics were largely unaltered suggested that compromised biliary clearance of CP-671,305 was compensated by increased urinary clearance. Overall, these studies suggest that hepatic transporters play an important role in the disposition and clearance of CP-671,305 in rat and human, and as such, these studies should aid in the design of clinical drug-drug interaction studies.