OATP1B1-mediated Transport Research Articles

Long-Lasting Inhibitory Effect of Apple and Orange Juices, but Not Grapefruit Juice, on OATP2B1-Mediated Drug Absorption

Enzyme-based grapefruit juice (GFJ)-drug interactions are mainly due to mechanism-based irreversible inhibition of metabolizing enzyme CYP3A4 by GFJ components, but the transporter organic anion transporting polypeptide (OATP)2B1 is also a putative site of interaction between drugs and fruit juices (FJ) in the absorption process. Here we aimed to investigate the effect of preincubation with FJ on OATP2B1-mediated transport of drugs in vitro. When OATP2B1-expressing Xenopus oocytes were preincubated with GFJ, orange juice (OJ), or apple juice (AJ), AJ induced a remarkable decrease in OATP2B1-mediated estrone-3-sulfate uptake in a concentration-dependent manner (IC(50) = 1.5%). A similar but less potent effect was observed with OJ (IC(50) = 21%), whereas GFJ had no effect. Similar results were obtained in preincubation studies using fexofenadine. Preincubation with OJ and AJ resulted in time-dependent inhibition of OATP2B1. Again, AJ had the more potent effect; its action lasted for at least 240 minutes, suggesting that AJ irreversibly inhibits OATP2B1-mediated drug uptake. Kinetic analysis revealed that coincubation and preincubation with AJ reduced OATP2B1-mediated estrone-3-sulfate uptake via competitive and noncompetitive mechanisms, respectively. Thus, OATP2B1 is functionally impaired through both competitive and long-lasting inhibition mechanisms by AJ and OJ, but not GFJ. Interestingly, although GFJ but not AJ is able to irreversibly inhibit CYP3A4, in the case of OATP2B1, AJ but not GFJ has a long-lasting inhibitory effect. Accordingly, complex FJ-drug interactions may occur in vivo, and their clinical significance should be examined.

Drug-Drug Interactions between Rosuvastatin and Oral Antidiabetic Drugs Occurring at the Level of OATP1B1

Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the V(max) for OATP1B1-mediated transport of E(2)17β-G (estradiol 17β-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 μM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17β-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

Influence of SLCO1B1 Polymorphisms on the Drug‐Drug Interaction Between Darunavir/Ritonavir and Pravastatin

The authors investigated whether SLCO1B1 polymorphisms contribute to variability in pravastatin pharmacokinetics when pravastatin is administered alone versus with darunavir/ritonavir. HIV-negative healthy participants were prospectively enrolled on the basis of SLCO1B1 diplotype: group 1 (*1A/*1A, n = 9); group 2 (*1A/*1B, n = 10; or *1B/*1B, n = 2); and group 3 (*1A/*15, n = 1; *1B/*15, n = 5; or *1B/*17, n = 1). Participants received pravastatin (40 mg) daily on days 1 through 4, washout on days 5 through 11, darunavir/ritonavir (600/100 mg) twice daily on days 12 through 18, with pravastatin 40 mg added back on days 15 through 18. Pharmacokinetic studies were conducted on day 4 (pravastatin alone) and day 18 (pravastatin + darunavir/ritonavir). Pravastatin area under the plasma concentration-time curve (AUC(tau)) was 21% higher during administration with darunavir/ritonavir compared with pravastatin alone; however, this difference was not statistically significant (P = .11). Group 3 variants had 96% higher pravastatin AUC(tau) on day 4 and 113% higher pravastatin AUC(tau) on day 18 compared with group 1. The relative change in pravastatin pharmacokinetics was largest in group 3 but did not differ significantly between diplotype groups. In sum, the influence of SLCO1B1*15 and *17 haplotypes on pravastatin pharmacokinetics was maintained in the presence of darunavir/ritonavir. Because OATP1B1 inhibition would be expected to be greater in carriers of normal or high-functioning SLCO1B1 haplotypes, these findings suggest that darunavir/ritonavir is not a potent inhibitor of OATP1B1-mediated pravastatin transport in vivo.

Steroid hormones specifically modify the activity of organic anion transporting polypeptides

Previously, the steroid hormone progesterone has been demonstrated to stimulate OATP2B1-mediated transport of estrone-3-sulphate (E1S), dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PS), which may influence the uptake of precursor molecules for steroid hormone synthesis. However, it is unclear whether OATP2B1 drug substrates like atorvastatin or glibenclamide are also affected by this phenomenon. In addition, it has not been studied so far if this stimulatory effect is specific for OATP2B1. To address these questions, we examined the influence of progesterone on OATP2B1-mediated atorvastatin and glibenclamide uptake and studied the impact of steroid hormones on the transport activity of OATP1A2, OATP1B1 and OATP1B3.Comparison of the substrate spectrum of the investigated OATPs revealed that DHEAS and atorvastatin are substrates of all transporters, while E1S was only significantly transported by OATP1A2, OATP2B1 and OATP1B1. Glibenclamide uptake was limited to OATP1A2, OATP1B1 and OATP2’B1.Subsequent interaction studies indicated that progesterone only increases OATP2B1-mediated E1S and DHEAS transport, whereas uptake of BSP, atorvastatin and glibenclamide was either inhibited or not affected. Moreover, the steroid hormone effect was specific for OATP2B1; neither OATP1B1, OATP1B3 nor OATP1A2 function was stimulated in the presence of progesterone. Similar to progesterone, the glucocorticoide dexamethasone stimulated OATP2B1-mediated transport of E1S and DHEAS (EC50 for E1S: 10.2±5.6μM and 17.9±15.4μM for DHEAS).In conclusion, our data demonstrate that among the tested compounds the stimulatory effect of progesterone is specific for OATP2B1 and restricted to sulphated steroids like E1S and DHEAS while the OATP-mediated drug transport is not enhanced.

Interactions of bilastine, a new oral H1 antihistamine, with human transporter systems

Membrane transporters play a significant role in facilitating transmembrane drug movement. For new pharmacological agents, it is important to evaluate potential interactions (e.g., substrate specificity and/or inhibition) with human transporters that may affect their pharmacokinetics, efficacy, or toxicity. Bilastine is a new nonsedating H1 antihistamine indicated for the treatment of allergic rhinoconjunctivitis and urticaria. The in vitro inhibitory effects of bilastine were assessed on 12 human transporters: four efflux [multidrug resistance protein 1 (MDR1) or P-glycoprotein, breast cancer resistance protein (BCRP), multidrug resistance associated protein 2 (MRP2), and bile salt export pump) and eight uptake transporters (sodium taurocholate cotransporting polypeptide, organic cation transporter (OCT)1, organic anion transporter (OAT)1, OAT3, OCT2, OATP2B1, OATP1B1, and OATP1B3). Only mild inhibition was found for MDR1-, OCT1-, and OATP2B1-mediated transport of probe substrates at the highest bilastine concentration assayed (300 μM; half-maximal inhibitory concentration: ≥300 μM). Bilastine transport by MDR1, BCRP, OAT1, OAT3, and OCT2 was also investigated in vitro. Only MDR1 active transport of bilastine was relevant, whereas it did not appear to be a substrate of OCT2, OAT1, or OAT3, nor was it transported substantially by BCRP. Drug-drug interactions resulting from bilastine inhibition of drug transporters that would be generally regarded as clinically relevant are unlikely. Additionally, bilastine did not appear to be a substrate of human BCRP, OAT1, OAT3, or OCT2 and thus is not a potential victim of inhibitors of these transporters. On the other hand, based on in vitro evaluation, clinically relevant interactions with MDR1 inhibitors are anticipated.

Prediction of the in vivo OATP1B1-mediated drug–drug interaction potential of an investigational drug against a range of statins

To support drug development, the drug–drug interaction potential (DDI) of an investigational drug (AZX) was assessed against the probe estradiol 17β-glucuronide as well as against simvastatin acid, atorvastatin, pravastatin, pitavastatin, fluvastatin, rosuvastatin and estrone 3-sulfate. The inhibitory potentials of the OATP1B1 inhibitors rifamycin SV and gemfibrozil were assessed in parallel. Monolayer cellular uptake assays were used to determine inhibition of human OATP1B1. Apparent Km values for the OATP1B1-mediated transport of [3H] substrates were determined prior to their use as probes in inhibition studies, and ranged from 0.6 to 29μM for statins. The Km of lipophilic simvastatin acid could not be determined due to its high passive permeability that masked OATP1B1 transport, and therefore this statin could not be used as a probe. Estrone 3-sulfate exhibited biphasic kinetics, whereas estradiol 17β-glucuronide demonstrated simple Michaelis–Menton kinetics. AZX moderately inhibited OATP1B1-mediated transport of all statins (IC50=4.6–9.7μM), except fluvastatin, of estradiol 17β-glucuronide (IC50=5.3μM), and weakly inhibited estrone 3-sulfate (IC50=79μM). Rifamycin SV strongly, and gemfibrozil weakly, inhibited the OATP1B1-mediated transport of substrates. Estradiol 17β-glucuronide was identified as a good surrogate probe for statins when assessing OATP1B1 inhibitory potential using this test system. Inhibition data was used to predict the likelihood of a clinical DDI, using current draft US FDA guidance and recommendations of the International Transporter Consortium. Predictions for AZX indicated the potential for an OATP1B1-mediated DDI in vivo and that a clinical interaction study is warranted to confirm whether AZX is an OATP1B1 inhibitor in the clinic.

Abstract 1848: Expression of an androgen transporter, organic anion transporting polypeptide 1B3, impacts progression and treatment of prostate cancer

Abstract Organic anion transporting polypeptides (OATPs) are membrane transporters that facilitate the movement of large substrates into cells. One family member, organic anion transporting polypeptide 1B3 (OATP1B3) is aberrantly overexpressed in prostate cancer and has been shown to influx both endogenous small molecules and chemotherapy reagents. Our hypothesis is that the OATP1B3 affects tumor growth by influxing testosterone. However, little is known about the cause of OATP1B3 expression in cancer and the impact of OATP1B3 transport on tumor growth. Therefore, we completed a study investigating putative hypoxia elements in the OATP1B3 promoter region, characterizing the impact of OATP1B3 androgen transport on prostate cancer growth, and examining the effect of OATP1B3 on the efficacy of standard prostate cancer treatments. The results showed that OATP1B3 co-localized with HIF 1-alpha in prostate cancer tissues from patients, correlated significantly to HIF1A in xenograft tumors under castration (P=0.005), and confirmed HIF-1alpha elements in the SLCO1B3 promoter region. In addition, radioactive transport assays with Xenopus oocytes showed that OATP1B3 transports testosterone and dihydrotestosterone 50- to 250-fold over passive diffusion at low physiological concentrations. A prostate cancer cell line expressing OATP1B3 grew faster than untransfected in response to androgens at all concentrations. Finally, OATP1B3 increased cell sensitivity to docetaxel treatment. In summary, these data show that hypoxia and castration increases OATP1B3 expression in cancer, OATP1B3-mediated androgen transport significantly impacts prostate cancer growth and OATP1B3 impacts the efficacy of docetaxel. These data provides evidence that OATP1B3 impacts prostate cancer growth and treatment. Future studies could screen for specific inhibitors of OATP1B3-mediated androgen transport. 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 1848. doi:1538-7445.AM2012-1848

Inhibition of the organic anion‐transporting polypeptide 1B1 by quercetin: an in vitro and in vivo assessment

To investigate the effect of quercetin on organic anion transporting polypeptide 1B1 (OATP1B1) activities in vitro and on the pharmacokinetics of pravastatin, a typical substrate for OATP1B1 in healthy Chinese-Han male subjects. Using human embryonic kidney 293 (HEK293) cells stably expressing OATP1B1, we observed the effect of quercetin on OATP1B1-mediated uptake of estrone-3-sulphate (E3S) and pravastatin. The influence of quercetin on the pharmacokinetics of pravastatin was measured in 16 healthy Chinese-Han male volunteers receiving a single dose of pravastatin (40 mg orally) after co-administration of placebo or 500 mg quercetin capsules (once daily orally for 14 days). Quercetin competitively inhibited OATP1B1-mediated E3S uptake with a K(i) value of 17.9 ± 4.6 µm and also inhibited OATP1B1-mediated pravastatin uptake in a concentration dependent manner (IC(50) , 15.9 ± 1.4 µm). In healthy Chinese-Han male subjects, quercetin increased the pravastatin area under the plasma concentration - time curve (AUC(0,10 h) and the peak plasma drug concentration (C(max)) to 24% (95% CI 15, 32%, P < 0.001) and 31% (95% CI 20, 42%, P < 0.001), respectively. After administration of quercetin, the elimination half-life (t(1/2) ) of pravastatin was prolonged by 14% (95% CI 4, 24%, P = 0.027), with no change in the time to reach C(max) (t(max) ). Moreover, quercetin decreased the apparent clearance (CL/F) of pravastatin by 18% (95% CI 75, 89%, P < 0.001). These findings suggest that quercetin inhibits the OATP1B1-mediated transport of E3S and pravastatin in vitro and also has a modest inhibitory influence on the pharmacokinetics of pravastatin in healthy Chinese-Han male volunteers. The effects of quercetin on other OATP1B1 substrate drugs deserve further investigation.

Inhibition of hepatic uptake transporters by flavonoids

Members of the human SLC superfamily such as organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, and organic cation transporter 1 (OCT1) are drug uptake transporters that are localised on the basolateral membrane of hepatocytes mediating the uptake of drugs such as atorvastatin and metformin into hepatocytes. Ingredients of food such as flavonoids influence the effects of drugs, e.g. by inhibition of drug transporters. Therefore, we investigated the impact of the Ginkgo biloba flavonoids apigenin, kaempferol, and quercetin, and the grapefruit flavonoids naringenin, naringin, and rutin on the OATP1B1, OATP1B3, and OCT1 transport activity. Transporter expressing HEK293 cell lines were used with [3H]sulfobromophthalein ([3H]BSP) as substrate for OATP1B1 and OATP1B3, [3H]atorvastatin as substrate for OATP1B1, and [3H]1-methyl-4-phenylpyridinium ([3H]MPP+) as substrate for OCT1. The G. biloba flavonoids showed a competitive inhibition of the OATP1B1- and OATP1B3-mediated [3H]BSP and the OATP1B1-mediated [3H]atorvastatin uptake. Quercetin was the most potent inhibitor of the OATP1B1- and OATP1B3-mediated [3H]BSP transport with Ki-values of 8.8±0.8μM and 7.8±1.7μM, respectively. For the inhibition of the OATP1B1-mediated [3H]atorvastatin transport, apigenin was the most potent inhibitor with a Ki value of 0.6±0.2μM. Among the grapefruit flavonoids, naringenin was the most potent inhibitor of the OATP1B1- and OATP1B3-mediated [3H]BSP transport with IC50-values of 81.6±1.1μM and 101.1±1.1μM, respectively. All investigated flavonoids showed no significant inhibition of the OCT1-mediated [3H]MPP+ uptake. Taken together, these in vitro studies showed that the investigated flavonoids inhibit the OATP1B1- and OATP1B3-mediated drug transport, which could be a mechanism for food–drug interactions in humans.

Substrate- and pH-Specific Antifolate Transport Mediated by Organic Anion-Transporting Polypeptide 2B1 (OATP2B1-SLCO2B1)

Human organic anion-transporting polypeptide (OATP) 2B1 (OATP-B; SLCO2B1) is expressed in the apical membrane of the small intestine and the hepatocyte basolateral membrane and transports structurally diverse organic anions with a wide spectrum of pH sensitivities. This article describes highly pH-dependent OATP2B1-mediated antifolate transport and compares this property with that of sulfobromophthalein (BSP), a preferred OATP2B1 substrate. At pH 5.5 and low substrate concentrations (~2.5 μM), only [(3)H]pemetrexed influx [in contrast to methotrexate (MTX), folic acid, and reduced folates] could be detected in OATP2B1-transfected HeLa R1-11 cells that lack endogenous folate-specific transporters. Influx was optimal at pH 4.5 to 5.5, falling precipitously with an increase in pH >6.0; BSP influx was independent of pH. Influx of both substrates at low pH was markedly inhibited by the proton ionophore 4-(trifluoromethoxy)phenylhydrazone; BSP influx was also suppressed at pH 7.4. At 300 μM MTX, influx was one-third that of pemetrexed; influx of folic acid, (6S)5-methyltetrahydrofolate, or (6S)5-formyltetrahydrofolate was not detected. There were similar findings in OATP2B1-expressing Xenopus laevis oocytes. The pemetrexed influx K(m) was ~300 μM; the raltitrexed influx K(i) was ~70 μM at pH 5.5. Stable expression of OAPT2B1 in HeLa R1-11 cells resulted in substantial raltitrexed, but modest pemetrexed, growth inhibition consistent with their affinities for this carrier. Hence, OATP2B1 represents a low-affinity transport route for antifolates (relative affinities: raltitrexed > pemetrexed > MTX) at low pH. In contrast, the high affinity of this transporter for BSP relative to antifolates seems to be intrinsic to its binding site and independent of the proton concentration.

In Vitro and In Silico Strategies to Identify OATP1B1 Inhibitors and Predict Clinical Drug–Drug Interactions

PurposeTo establish in vitro and in silico models that predict clinical drug–drug interactions (DDIs) with the OATP1B1 (SLCO1B1) transporter.MethodsThe inhibitory effect of 146 drugs and drug-like compounds on OATP1B1-mediated transport was studied in HEK293 cells. A computational model was developed to predict OATP1B1 inhibition. Concentration-dependent effects were investigated for six compounds; clinical DDIs were predicted by calculating change in exposure (i.e. R-values) in eight different ways.ResultsSixty-five compounds were identified as OATP1B1 inhibitors at 20 μM. The computational model predicted the test set with 80% accuracy for inhibitors and 91% for non-inhibitors. In vitro–in vivo comparisons underscored the importance of using drugs with known clinical effects as references. Thus, reference drugs, cyclosporin A, gemfibrozil, and fenofibrate, provided an inhibition interval to which three antiviral drugs, atazanavir, lopinavir, and amprenavir, could be compared and their clinical DDIs with OATP1B1 classified.ConclusionsTwenty-two new OATP1B1 inhibitors were identified, a predictive OATP1B1 inhibition in silico model was developed, and successful predictions of clinical DDIs were obtained with OATP1B1.

Isolation of Modulators of the Liver-Specific Organic Anion-Transporting Polypeptides (OATPs) 1B1 and 1B3 from Rollinia emarginata Schlecht (Annonaceae)

Organic anion-transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are liver-specific transporters that mediate the uptake of a broad range of drugs into hepatocytes, including statins, antibiotics, and many anticancer drugs. Compounds that alter transport by one or both of these OATPs could potentially be used to target drugs to hepatocytes or improve the bioavailability of drugs that are cleared by the liver. In this study, we applied a bioassay-guided isolation approach to identify such compounds from the organic extract of Rollinia emarginata Schlecht (Annonaceae). Fractions of the plant extract were screened for effects on OATP1B1- and OATP1B3-mediated transport of the model substrates estradiol-17β-glucuronide and estrone-3-sulfate. We isolated three compounds, ursolic acid, oleanolic acid, and 8-trans-p-coumaroyloxy-α-terpineol, which inhibited estradiol-17β-glucuronide uptake by OATP1B1 but not OATP1B3. In addition, a rare compound, quercetin 3-O-α-l-arabinopyranosyl(1→2) α-L-rhamnopyranoside, was identified that had distinct effects on each OATP. OATP1B1 was strongly inhibited, as was OATP1B3-mediated transport of estradiol-17β-glucuronide. However, OATP1B3-mediated uptake of estrone-3-sulfate was stimulated 4- to 5-fold. Kinetic analysis of this stimulation revealed that the apparent affinity for estrone-3-sulfate was increased (decreased K(m)), whereas the maximal rate of transport (V(max)) was significantly reduced. These results demonstrate a mechanism through which the hepatic uptake of drug OATP substrates could be stimulated.

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.

Influence of Non-Steroidal Anti-Inflammatory Drugs on Organic Anion Transporting Polypeptide (OATP) 1B1- and OATP1B3-Mediated Drug Transport

The transporter-mediated uptake of drugs from blood into hepatocytes is a prerequisite for intrahepatic drug action or intracellular drug metabolism before excretion. Therefore, uptake transporters, e.g., members of the organic anion transporting polypeptide (OATP) family are important determinants of drug pharmacokinetics. Highly and almost exclusively expressed in hepatocytes are the OATP family members OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3). Drug substrates of OATP1B1 and OATP1B3 include antibiotics and HMG-CoA reductase inhibitors (statins). It has been demonstrated that administration of two or more drugs that are substrates for these hepatic uptake transporters may lead to transporter-mediated drug-drug interactions, resulting in altered transport kinetics for drug substrates. In this study we investigated whether non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol interact with OATP1B1 and OATP1B3 using the standard substrate BSP and the drug substrate pravastatin. Using human embryonic kidney cells stably expressing OATP1B1 or OATP1B3, we demonstrated that bromosulfophthalein uptake was inhibited by diclofenac, ibuprofen. and lumiracoxib. Of interest, pravastatin uptake was stimulated by these NSAIDs, and for ibuprofen we determined activation constants (EC₅₀ values) of 64.0 and 93.1 μM for OATP1B1- and OATP1B3-mediated uptake, respectively. Furthermore, we investigated whether NSAIDs were also substrates for OATP1B1 and OATP1B3 and demonstrated that only diclofenac was significantly transported by OATP1B3, whereas all other NSAIDs investigated were not substrates for these uptake transporters. These results demonstrated that drugs may interact with transport proteins by allosteric mechanisms without being substrates and, therefore, not only uptake inhibition but also allosteric-induced modulation of transport function may be an important mechanism in transporter-mediated drug-drug interactions.

Inhibitory effects of ketoconazole and rifampin on OAT1 and OATP1B1 transport activities: considerations on drug–drug interactions

Ketoconazole and rifampin are the most widely used compounds examined in recent drug-drug interaction (DDI) studies, and they have multiple roles in modulating drug metabolizing enzymes and transporters. To determine the underlying mechanisms of DDI, this study was performed to investigate the inhibitory effects of ketoconazole and rifampin on the functions of OAT1 and OATP1B1, and to evaluate the potential of ketoconazole and rifampin for DDI with substrate drugs for these transporters in a clinical setting. Ketoconazole inhibited OATP1B1-mediated transport activity, while rifampin inhibited OAT1 and OATP1B1. Inhibition by rifampin and ketoconazole of the uptake of olmesartan, a substrate for OAT1 and OATP1B1, was evaluated in oocytes overexpressing these transporters. The K(i) values for rifampin on OAT1 and OATP1B1-mediated olmesartan uptake were 62.2 and 4.42 µM, respectively, and the K(i) value for ketoconazole on OATP1B1-mediated olmesartan uptake was 66.1 µM. As measured plasma concentrations of rifampin and ketoconazole were 7.29 and 6.4-13.3 µM, respectively, the likelihood of an OATP1B1-mediated drug-drug interaction between rifampin and olmesartan is thought to be possible, whereas OAT1 or OATP1B1-mediated DDI between rifampin or ketoconazole and olmesartan appears unlikely in the clinical setting.

Screening of Antibiotics That Interact with Organic Anion-Transporting Polypeptides 1B1 and 1B3 Using Fluorescent Probes

Hepatic organic anion transporters OATP1B1 and OATP1B3 are expressed at the sinusoidal membrane of hepatocytes and contribute to the hepatic uptake of a wide variety of clinically used drugs. To identify the antibiotics that interact with the human organic anion transporters OATP1B1 and OATP1B3, we applied a screening system using fluorescent probes. Twenty-six antibiotics with a variety of mechanisms of action were examined. The screening demonstrated that four antibiotics inhibited OATP1B1-mediated transport and 11 antibiotics inhibited OATP1B3-mediated transport in a concentration-dependent manner. Antibiotics that inhibited OATP1B3-mediated transport tended to exhibit higher affinity than those that inhibited OATP1B1-mediated transport. To clarify whether the antibiotics that interacted with OATP1B1 and/or OATP1B3 were substrates for these transporters, an uptake study was performed. Rifampicin and penicillin were transported by both OATP1B1 and OATP1B3. Moreover, OATP1B3 was involved in the transport of ceftriaxone, cefmetazole, cefoperazone, and cefotaxime. Macrolides were not significantly transported by either transporter. In conclusion, the results demonstrated that our system is a useful method for the rapid screening of transporter-antibiotic interaction, and we found novel substrates. Our results indicate that OATP1B1 and/or OATP1B3 contribute to the transport process of some antibiotics, and that drug-drug interactions associated with these transporters could occur after the administration of antibiotics.

Influence of the flavonoids apigenin, kaempferol, and quercetin on the function of organic anion transporting polypeptides 1A2 and 2B1

OATP1A2 and OATP2B1 are uptake transporters of the human organic anion transporting polypeptide (OATP) family with a broad substrate spectrum including several endogenous compounds as well as drugs such as the antihistaminic drug fexofenadine and HMG-CoA reductase inhibitors. Both transporters are localized in the apical membrane of human enterocytes. Flavonoids, abundantly occurring in plants, have previously been shown to interact with drug metabolizing enzymes and transporters. However, the impact of flavonoids on OATP1A2 and OATP2B1 transport function has not been analyzed in detail. Therefore, HEK293 cell lines stably expressing OATP1A2 and OATP2B1 were used to investigate the influence of the Ginkgo flavonoids apigenin, kaempferol, and quercetin on the transport activity of OATP1A2 and OATP2B1. K i values of all three flavonoids determined from Dixon plot analyses using BSP as substrate indicated a competitive inhibition with quercetin as the most potent inhibitor of OATP1A2 (22.0 μM) and OATP2B1 (8.7 μM) followed by kaempferol (OATP1A2: 25.2 μM, OATP2B1: 15.1 μM) and apigenin (OATP1A2: 32.4 μM OATP2B1: 20.8 μM). Apigenin, kaempferol, and quercetin led to a concentration-dependent decrease of the OATP1A2-mediated fexofenadine transport with IC 50 values of 4.3 μM, 12.0 μM, and 12.6 μM, respectively. The OATP1A2- and OATP2B1-mediated transport of atorvastatin was also efficiently inhibited by apigenin (IC 50 for OATP1A2: 9.3 μM, OATP2B1: 13.9 μM), kaempferol (IC 50 for OATP1A2: 37.3 μM, OATP2B1: 20.7 μM) and quercetin (IC 50 for OATP1A2: 13.5 μM, OATP2B1: 14.1 μM). These data indicate that modification of OATP1A2 and OATP2B1 transport activity by apigenin, kaempferol, and quercetin may be a mechanism for food–drug or drug–drug interactions in humans.

Fluorescence-based assays for the assessment of drug interaction with the human transporters OATP1B1 and OATP1B3

Hepatic disposition plays a significant role in the pharmacokinetics and pharmacodynamics of a variety of drugs. Sinusoidal membrane transporters have been shown to participate in the hepatic disposition of many pharmaceuticals. Two sinusoidal membrane transporters with an established role in hepatic disposition are OATP1B1 and OATP1B3 (organic anion-transporting polypeptides 1B1 and 1B3, respectively). OATP1B1 and OATP1B3 have been implicated in the hepatic uptake of statin drugs, and polymorphisms linked to OATP1B1 have been associated with deleterious patient endpoints. As a result, OATP1B1 and OATP1B3 represent sites for potential drug–drug interactions. Numerous methods exist for identifying potential drug–drug interactions with transporters. However, relatively few offer the convenience and speed of fluorescence-based assays. Here a fluorescence-based assay was developed for measuring the OATP1B1- and OATP1B3-mediated transport of 8-fluorescein-cAMP (8-FcA). The OATP1B1- and OATP1B3-mediated transport of 8-FcA was time dependent and saturable ( K m = 2.9 and 1.8 μM, V max = 0.20 and 0.33 pmol/min/cm 2, respectively). Molecules known to interact with OATPs, including cyclosporin A, rifampicin, and glibenclamide, each demonstrated concentration-dependent inhibition of 8-FcA transport by OATP1B1 and OATP1B3. The in vitro fluorescence-based assays described here using 8-FcA as the substrate are convenient and rapid and have utility in screening drug candidates for potential drug–drug interactions with OATP1B1 and OATP1B3.

Cyclosporine A, but Not Tacrolimus, Shows Relevant Inhibition of Organic Anion-Transporting Protein 1B1-Mediated Transport of Atorvastatin

The aim of this study was to investigate the potential of calcineurin inhibitors [cyclosporine A (CsA) and tacrolimus (Tac)] to inhibit cellular uptake of atorvastatin mediated by the liver-specific organic anion-transporting polypeptide 1B1 (OATP1B1) in vitro. Patients with solid organ transplants are frequently treated with HMG-CoA reductase inhibitors (statins). CsA increases atorvastatin systemic exposure severalfold, an effect not observed with Tac. The effect of CsA and Tac on atorvastatin transport via OATP1B1 was investigated in transfected human embryonic kidney 293 cells. An in vitro-in vivo extrapolation (IVIVE) was performed to estimate the clinical potential for CsA and Tac to inhibit OATP1B1-mediated transport. CsA inhibited OATP1B1-mediated uptake of atorvastatin approximately 90-fold more efficiently than Tac, with half-maximal inhibitory concentration (IC(50)) values of 0.021 +/- 0.004 and 1.99 +/- 0.42 muM, respectively. Coincubation compared with preincubation with CsA showed a 20-fold lower inhibitory capacity, with an IC(50) value of 0.47 +/- 0.34 muM. The IVIVE showed that clinically obtainable concentrations of CsA, but not Tac, inhibit OATP1B1 transport of atorvastatin. CsA inhibition ranged from 28 to 77% within a dosing interval, whereas it was less than 1% for Tac, considering free concentrations and assuming competitive inhibition. This does not fully explain the clinically observed interaction with CsA, suggesting that a more complex inhibitory mechanism may be present. This is also supported by the decreased IC(50) value of CsA after preincubation. This study provides evidence that OATP1B1 inhibition is a relevant mechanism for the interaction observed between CsA and atorvastatin.

PH Dependence of Organic Anion-Transporting Polypeptide 2B1 in Caco-2 Cells: Potential Role in Antiretroviral Drug Oral Bioavailability and Drug–Drug Interactions

Human intestinal epithelium expresses a number of drug efflux and influx transporters that can restrict and/or facilitate intestinal drug uptake during absorption. Organic anion-transporting polypeptide 2B1 (OATP2B1), a multispecific organic anion uptake transporter localized at the brush-border membrane of intestinal epithelial cells, is known to transport many endogenous substrates (e.g., steroid conjugates) and xenobiotics (e.g., statins). At present, limited information is available on the mechanism of HIV protease inhibitor (PIs) intestinal uptake. In this study, we examined the interaction of PIs with the OATP2B1 transport system in Caco-2 cells, an in vitro model of human intestinal epithelium, and Madin-Darby canine kidney II cells stably transfected with OATP2B1. The expression of OATP2B1 transcript and protein was confirmed by reverse transcription-polymerase chain reaction and immunoblot analysis, respectively. Estrone-3-sulfate (E3S) uptake demonstrated biphasic saturation kinetics in Caco-2 cells, with dissociation constants (K(M)) of 6 +/- 2 microM and 1.5 +/- 0.2 mM. Several PIs potently inhibited OATP2B1-mediated transport in Caco-2 cells at clinically relevant IC(50) concentrations for ritonavir (0.93 microM), atazanavir (2.2 microM), lopinavir (1.7 microM), tipranavir (0.77 microM), and nelfinavir (2.2 microM). An inwardly directed proton gradient was identified as the driving force of E3S uptake through NH(4)Cl intracellular acidification studies with a H(+):E3S stoichiometry for OATP2B1 of 1:1. In contrast, although atazanavir and ritonavir uptake by Caco-2 cells was stimulated by low extracellular pH, this process was not mediated by OATP2B1 and was not affected by an outwardly directed H(+) gradient. Because OATP2B1 exhibits an increasing number of drug substrates, including several statins, alterations of its function by PIs could result in clinically significant drug-drug interactions in the intestine.