Organic Anion Transporting Polypeptide Substrates Research Articles

Structural determination of OATP transporters utilising homology models and cell based assays

Membrane transport proteins (transporters) are present in the lipid bilayer of cells and selectively transport compounds across the membrane. The organic anion transporting polypeptides (OATPs) are a family of membrane transporters expressed in a variety of tissues, including absorptive/excretory cells of the liver, kidney and intestine. In addition, OATPs transport an array of endogenous and xenobiotic compounds including bile salts, thyroid hormones, statins and antineoplastics. OATPs play an important role in cell homeostasis and drug absorption/excretion, and have also been implicated in disease and drug‐drug interactions. However there is still much to determine regarding the binding properties and exact structure of these proteins. The topology and binding properties of OATP1A2, OATP1B1, OATP1B3 and OATP2B1 have been investigated using a HEK293T cell model.OATP proteins have been expressed in HEK293T cells and kinetic properties determined using the OATP substrate estrone‐3‐sulfate. Bioinformatics analysis using various topology models has revealed that OATP1B1, OATP1B3 and OATP2B1 have between 11–12 transmembrane domains. However there could be as few as 8 domains, as is the predicted topology of the less well characterised OATP1A2. The topology of these proteins has been experimentally investigated using a FLAG epitope tag recognition system. Epitope tags were cloned into the putative extracellular regions of the protein, which were recognised by a FLAG antibody. The detection of the antibody was quantified by a chemiluminescence method and those detected confirmed as extracellular regions. The findings of this study will be used to further determine drug transport pathways and aid in future drug development.

Mechanistic Pharmacokinetic Modeling for the Prediction of Transporter-Mediated Disposition in Humans from Sandwich Culture Human Hepatocyte Data

With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic anion-transporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under- and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.

OATPs, OATs and OCTs: the organic anion and cation transporters of the SLCO and SLC22A gene superfamilies

The human organic anion and cation transporters are classified within two SLC superfamilies. Superfamily SLCO (formerly SLC21A) consists of organic anion transporting polypeptides (OATPs), while the organic anion transporters (OATs) and the organic cation transporters (OCTs) are classified in the SLC22A superfamily. Individual members of each superfamily are expressed in essentially every epithelium throughout the body, where they play a significant role in drug absorption, distribution and elimination. Substrates of OATPs are mainly large hydrophobic organic anions, while OATs transport smaller and more hydrophilic organic anions and OCTs transport organic cations. In addition to endogenous substrates, such as steroids, hormones and neurotransmitters, numerous drugs and other xenobiotics are transported by these proteins, including statins, antivirals, antibiotics and anticancer drugs. Expression of OATPs, OATs and OCTs can be regulated at the protein or transcriptional level and appears to vary within each family by both protein and tissue type. All three superfamilies consist of 12 transmembrane domain proteins that have intracellular termini. Although no crystal structures have yet been determined, combinations of homology modelling and mutation experiments have been used to explore the mechanism of substrate recognition and transport. Several polymorphisms identified in members of these superfamilies have been shown to affect pharmacokinetics of their drug substrates, confirming the importance of these drug transporters for efficient pharmacological therapy. This review, unlike other reviews that focus on a single transporter family, briefly summarizes the current knowledge of all the functionally characterized human organic anion and cation drug uptake transporters of the SLCO and the SLC22A superfamilies.

Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β- d-glucuronide (E 2-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly ( p < 0.01) decreased the accumulation of estrone-3-sulfate and E 2-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant ( p < 0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation ( R 2 = 0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Organic Anion Transporting Polypeptides (OATPs): Regulation of Expression and Function

Eleven members of the human organic anion transporter (OATP) family (grouped into six families) facilitate the Na(+)- independent transmembrane transport of various endo- and xenobiotics (bile acids, bilirubin, steroid hormone conjugates, thyroid hormones, prostaglandins, clinically used drugs, and toxins). OATPs are 12-transmembrane glycoproteins (643-722 amino acids) and contain many conserved structural features, for example, eleven cysteines in the large extracellular loop 5. They are important for proper transport, for which translocation of substrates through a central, positively-charged pore in a rocker-switch-type mechanism has been proposed. Although OATPs are expressed in various cells and tissues, some members show a more restricted pattern (well-studied OATP1B1/OATP1B3 in liver, OATP4C1 in kidney, and OATP6A1 in testis). In cancer, the distribution pattern is no longer maintained, and OATPs, like OATP1B3, become upregulated in malignant tissues (colon, breast, prostate). Studies in cell lines and animal models further revealed that the expression of OATPs is regulated in a cell- and tissue-specific way by cytokines and activation of nuclear receptors (LXR, FXR, PXR, CAR, HNF4). Also epigenetic mechanisms and postranslational modifications influence their expression and function. Therefore, changes in the expression of OATPs under pathological conditions will influence transport processes causing an altered accumulation of OATP substrates in cells of excretory organs (intestine, liver, kidney) and on various blood/organ barriers (such as brain, testis, placenta). For drugs, this may result in increased toxicity and adverse drug reactions. Therefore, it is important to improve the knowledge on the regulation and function of individual OATPs, and to apply it for therapeutic considerations.

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.

Organic anion transporting polypeptide 1a/1b–knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.

Inhibition of Hepatic Organic Anion-Transporting Polypeptide by RNA Interference in Sandwich-Cultured Human Hepatocytes: An In Vitro Model to Assess Transporter-Mediated Drug-Drug Interactions

Organic anion-transporting polypeptides (OATPs), members of the SLCO/SLC21 family, mediate the transport of various endo- and xenobiotics. In human liver, OATP1B1, 1B3, and 2B1 are located at the basolateral membrane of hepatocytes and are involved in hepatic drug uptake and biliary elimination. Clinically significant drug-drug interactions (DDIs) mediated by hepatic OATPs have drawn great attention from clinical practitioners and researchers. However, there are considerable challenges to prospectively understanding the extent of OATP-mediated DDIs because of the lack of specific OATP inhibitors or substrates and the limitations of in vitro tools. In the present study, a novel RNA interference knockdown sandwich-cultured human hepatocyte model was developed and validated. Quantitative polymerase chain reaction, microarray and immunoblotting analyses, along with uptake assays, illustrated that the expression and transport activity of hepatic OATPs were reduced by small interfering (siRNA) efficiently and specifically in this model. Although OATP siRNA decreased only 20 to 30% of the total uptake of cerivastatin into human hepatocytes, it caused a 50% reduction in cerivastatin metabolism, which was observed by monitoring the formation of the two major metabolites of cerivastatin. The results suggest that coadministration of a drug that is a hepatic OATP inhibitor could significantly alter the pharmacokinetic profile of cerivastatin in clinical studies. Further studies with this novel model demonstrated that OATP and cytochrome P450 have a synergistic effect on cerivastatin-gemfibrozil interactions. The siRNA knockdown sandwich-cultured human hepatocytes may provide a new powerful model for evaluating DDIs.

The role of organic anion transporting polypeptides (OATPs/ SLCOs) in the toxicity of different microcystin congeners in vitro: A comparison of primary human hepatocytes and OATP-transfected HEK293 cells

Cellular uptake of microcystins (MCs), a family of cyclic cyanobacterial heptapeptide toxins, occurs via specific organic anion transporting polypeptides (OATPs), where MCs inhibit serine/threonine-specific protein phosphatase (PP). Despite comparable PP-inhibitory capacity, MCs differ greatly in their acute toxicity, thus raising the question whether this discrepancy results from MC-specific toxikokinetic rather than toxicodynamic differences. OATP-mediated uptake of MC congeners MCLR, -RR, -LW and –LF was compared in primary human hepatocytes and HEK293 cells stably expressing recombinant human OATP1B1/ SLCO1B1 and OATP1B3/ SLCO1B3 in the presence/absence of OATP substrates taurocholate (TC) and bromosulfophthalein (BSP) and measuring PP-inhibition and cytotoxicity. Control vector expressing HEK293 were resistant to MC cytotoxicity, while TC and BSP competition experiments reduced MC cytotoxicity in HEK293-OATP transfectants, thus confirming the requirement of OATPs for trans-membrane transport. Despite comparable PP-inhibiting capabilities, MCLW and -LF elicited cytotoxic effects at lower equimolar concentrations than MCLR and MCRR, hence suggesting congener selective transport into HEK293-OATP transfectants and primary human hepatocytes. Primary human hepatocytes appeared one order of magnitude more sensitive to MC congeners than the corresponding HEK293 -OATP transfectants. Although the latter maybe due to a much lower level of PPs in primary human hepatocytes, the presence of OATPs other than 1B1 or 1B3 may have added to an increased uptake of MCs. In view of the high sensitivity of human hepatocytes and currently MCLR-only based risk calculations, the actual risk of human MC-intoxication and ensuing liver damage could be underestimated in freshwater cyanobacterial blooms where MCLW and-LF predominate.

Interaction of HIV protease inhibitors with OATP1B1, 1B3, and 2B1

The effects of human immunodeficiency virus (HIV) protease inhibitors (PI) on the accumulation of the fluorescent bile salt analogue cholyl-glycylamido-fluorescein (CGamF) were determined in organic anion transporting polypeptide (OATP)-1B1 and -1B3-expressing Chinese hamster ovary (CHO) cells. In addition, interaction studies in Caco-2 monolayers, known only to express the OATP2B1 isoform, were conducted using the established OATP substrate estrone 3-sulfate (E3S), since no CGamF accumulation was observed in Caco-2 monolayers.CGamF appeared an excellent substrate for the OATP1B subfamily, with net accumulation clearance values of 7.8 and 142 μl min−1 mg−1 protein in OATP1B1 and OATP1B3-transfected cells, respectively. Ki-values reflecting inhibition of CGamF accumulation by HIV PI correlated well between OATP1B1 and OATP1B3-expressing cells. Lopinavir was the most potent inhibitor (Ki = 0.5–1.4 μM) of OATP1B-mediated CGamF accumulation compared with atazanavir, darunavir, ritonavir, and saquinavir (Ki between 1.4 and 3.3 μM).Inhibitory profiles towards OATP2B1-mediated E3S accumulation were different with only indinavir, saquinavir, and ritonavir showing substantial effects.In conclusion, OATP1B3 appears to be a major transport mechanism mediating sodium-independent CGamF accumulation in human liver, and CGamF could be used as a probe substrate for in vitro drug interaction studies. The remarkably potent inhibition of OATP1B1 by lopinavir may explain some clinically relevant drug interactions between lopinavir and OATP1B substrates such as fexofenadine.

Interaction 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) flavanols on the function of three OATPs expressed on the basolateral membrane of human hepatocytes. Uptake of the model substrate estrone‐3‐sulfate was measured using CHO cells stably expressing OATP1B1, OATP1B3, or OATP2B1 in the absence and presence of the four most abundant flavanols found in green tea. Uptake by 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 (5‐fold increase, EC50 = 10.9 μM). The effect of EGCG on OATP1B3 was also studied with additional substrates: uptake of estradiol‐17β‐glucuronide was not affected, while uptake of Fluo‐3 was inhibited (IC50 of 8.3μM). These results indicate that two of the major flavanols found in green tea have a substantial effect upon the function of OATPs expressed in hepatocytes and can potentially alter the pharmacokinetics of drugs and other OATP substrates. These substrate dependent effects of EGCG on OATP1B3 seem to affect different transport/binding sites. In the future, we will use LC‐MS/MS analysis to identify whether these compounds are directly transported by OATPs.

EVALUATION OF DRUG-DRUG INTERACTION IN THE HEPATOBILIARY AND RENAL TRANSPORT OF DRUGS

Recent studies have revealed the import role played by transporters in the renal and hepatobiliary excretion of many drugs. These transporters exhibit a broad substrate specificity with a degree of overlap, suggesting the possibility of transporter-mediated drug-drug interactions with other substrates. This review is an overview of the roles of transporters and the possibility of transporter-mediated drug-drug interactions. Among the large number of transporters, we compare the Ki values of inhibitors for organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs) and their therapeutic unbound concentrations. Among them, cephalosporins and probenecid have the potential to produce clinically relevant OAT-mediated drug-drug interactions, whereas cyclosporin A and rifampicin may trigger OATP-mediated ones. These drugs have been reported to cause drug-drug interactions in vivo with OATs or OATP substrates, suggesting the possibility of transporter-mediated drug-drug interactions. To avoid adverse consequences of such transporter-mediated drug-drug interactions, we need to be more aware of the role played by drug transporters as well as those caused by drug metabolizing enzymes.

The effects of fruit juices on drug disposition: a new model for drug interactions.

Grapefruit juice produces mechanism-based inhibition of intestinal drug metabolism when consumed in normal quantities. This can produce clinically important increases in oral drug bioavailability when coadministered with substrates of cytochrome p450 3A4 (CYP3A4) that undergo high presystemic metabolism. Furanocoumarins such as bergamottin and 6',7'-dihydroxybergamottin have been identified as probable active constituents. Grapefruit juice may also inhibit intestinal P-glycoprotein-mediated efflux transport of drugs such as cyclosporine to increase its oral bioavailability. However, grapefruit juice does not enhance the absorption of digoxin, a prototypical P-glycoprotein substrate, likely because it has high inherent oral bioavailability. Grapefruit and other fruit juices have recently been shown to be potent in vitro inhibitors of a number of organic anion-transporting polypeptides (OATPs). These juices were also found to decrease the absorption of the nonmetabolized OATP substrate, fexofenadine. Taken together, the data support inhibition of intestinal uptake transporters by fruit juices to decrease drug bioavailability. This would represent a new mechanism for food-drug interactions. These findings with grapefruit and other fruit juices continue to enhance our understanding of the complex nature of food-drug interactions, and their possible influence on the clinical effects of medications.

Active Influx Transport is Mediated by Members of the Organic Anion Transporting Polypeptide Family in Human Epidermal Keratinocytes

Normal human epidermal keratinocytes have been shown to express a cell-type-specific pattern of extrahepatic cytochrome P450 enzymes and efflux transport proteins showing that these cells metabolize and excrete a variety of xenobiotics. Recently transport proteins involved in the uptake of xenobiotics have been detected and here we analyzed the mRNA and protein expression profiles and functional activities of these proteins in human keratinocytes in comparison to primary liver cells. The transporters studied included the subtypes A, B, C, D, and E of the organic anion transporting polypeptide (OATP) family, which are responsible for the uptake of various anionic and neutral molecules and especially organic cations - including drugs. Constitutive expression of OATP-B, OATP-D, and OATP-E was shown for the first time in normal human epidermal keratinocytes on a molecular level using reverse transcription polymerase chain reaction and northern blot analysis, as well as in human skin tissue shown by tissue blot hybridization and immunohistochemistry. Expression of OATP-A and OATP-C was not detected in any of the keratinocyte samples. In contrast, liver tissue showed a significant expression of OATP-A and OATP-B as well as OATP-C, a weak expression of OATP-D, and no expression of OATP-E. These data revealed that normal human epidermal keratinocytes express a specific profile of transporters involved in drug influx. Using a newly developed uptake-transport assay, uptake of known and well-characterized OATP substrates like estradiol-17beta-glucuronide and estrone sulfate was inhibited in normal human epidermal keratinocytes by specific inhibitors such as taurocholate, verifying the functional capacity of the expressed OATPs. Human dermal fibroblasts seem to have a lower influx transport activity for estradiol-17beta-glucuronide, which correlates with the immunohistologic data. Even though the substrate specificity of the OATP isoforms is only partially known until now, our findings support the concept that uptake of large organic cations like drugs in keratinocytes is an active transport process mediated by members of the OATP family.

Pregnancy within 8 months of a previous birth was associated with an increased rate of preterm birth

Accurately predicting hepatic clearance is an integral part of the drug-development process, and yet current in vitro to in vivo (IVIVE) extrapolation methods yield poor predictions, particularly for highly protein-bound...

Multispecific amphipathic substrate transport by an organic anion transporter of human liver

Background: Hepatic uptake of differently charged amphipathic endo- and xenobiotics is thought to occur via distinct carrier-mediated transport systems. Alternatively, a single rat organic anion transporting polypeptide (oatp) has recently been demonstrated to mediate hepatocellular uptake of differently charged amphipathic substrates. Aim: To investigate whether a cloned human liver organic anion transporting polypeptide (OATP) also can mediate charge- and class-independent hepatocellular uptake of amphipathic substrates. Methods: Xenopus laevis oocytes were injected with OATP-cRNA. Sodium-independent uptake of estrone-3-sulfate, ouabain and the organic cation N-(4,4-azo-n-pentyl)-21-ajmalinium was compared in OATP-expressing and uninjected (or water injected) control oocytes. Results: Our results indicate that OATP, in addition to bromosulfophthalein and bile salts, can also trasnport anionic estrone-3-sulfate (KM≈59 μM), neutral ouabain ( K m≈5.5 mM) and cationic N-(4,4-azo-n-pentyl)-21-ajmalinium. For each of these compounds, OATP-mediated uptake was cis-inhibited by the OATP substrate taurochenodeoxycholate and the transport activities correlated well with the amounts of cRNA injected. Conclusion: Similar to the rat liver oatp, the human liver OATP can also mediate multispecific and charge-independent uptake of lipophilic amphipathic organic compounds. Thus, OATP may play an important role in the first pass clearance of drugs and other xenobiotics by the human liver.