Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (K_m and V_max) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.     

The Influence of Oral Antidiabetic Drugs on Cellular Drug Uptake Mediated by Hepatic OATP Family Members

As patients with type 2 diabetes receiving oral antidiabetic drugs are often concomitantly treated with other drugs, they are of increased risk for drug interactions. Drugs have to be taken up into hepatocytes before their intracellular drug action or before they are metabolized, and therefore, uptake transporters are important modulators of drug pharmacokinetics and drug effects. To gain more insights into the role of uptake transporters for drug interactions, we investigated whether frequently prescribed oral antidiabetic drugs interact with the transport of drugs, mediated by the hepatic uptake transporters OATP1B1 (gene symbol SLCO1B1), OATP1B3 (gene symbol SLCO1B3) and OATP2B1 (gene symbol SLCO2B1). Using HEK293 cells recombinantly over‐expressing these uptake transporters, we analysed whether glibenclamide, glimepiride, nateglinide and pioglitazone influence the transport of the model transport substrate bromosulfophthalein. Furthermore, we investigated the influence of the same oral antidiabetic drugs and of repaglinide and rosiglitazone on the uptake of the HMG‐CoA‐reductase inhibitor atorvastatin. The oral antidiabetic drugs glibenclamide, glimepiride and nateglinide inhibited the transport of the model substrate bromosulfophthalein, particularly the OATP2B1‐mediated uptake. The OATP‐mediated atorvastatin uptake was inhibited in a similar manner. For glibenclamide, inhibitory constants (K_i values) of 13.6 μM, 8.1 μM and 0.5 μM for OATP1B1‐, OATP1B3‐ and OATP2B1‐mediated BSP uptake were determined. In conclusion, these in vitro results demonstrate that several oral antidiabetic drugs may influence hepatic OATP‐mediated drug uptake. The in vivo consequences of these results have to be analysed in further studies.     

Functional characterization for polymorphic organic anion transporting polypeptides (OATP/SLCO1B1, 1B3, 2B1) of monkeys recombinantly expressed with various OATP probes

The hepatic uptake of clinical drugs mediated by human hepatic organic anion transporting polypeptides (OATP/SLCO) has been reported extensively. In this study, hepatic uptake by recombinantly expressed monkey OATP1B1, OATP1B3 and OATP2B1 was investigated using three human OATP1B1 and OATP1B3 substrates (pitavastatin, pravastatin and rosuvastatin) and one OATP1B3 substrate (telmisartan), as the governmental drug interaction guidelines recommend, and seven reported clinical drugs. The uptake of known human probes into recombinant OATP‐expressing cells was significantly greater than that of mock cells. Consequently, pitavastatin, pravastatin and rosuvastatin were suggested to be substrates of recombinant monkey OATP1B1 and OATP1B3, and telmisartan was suggested to be a substrate of recombinant monkey OATP1B3, in a manner similar to human OATPs. In contrast, atorvastatin, bosentan, etoposide, fexofenadine, fluvastatin, glibenclamide and simeprevir were broadly transported by recombinant monkey OATP1B1, OATP1B3 and OATP2B1. Furthermore, some of the 16 non‐synonymous monkey OATP1B1 variants found in 64 cynomolgus and 32 rhesus monkeys mediated up to a 1.6‐fold [³H]pitavastatin uptake (with low Michaelis constant values) in comparison with the wild type under the present conditions. Despite sequences of monkey recombinant OATPs not being totally reflective of those of human OATPs, our results collectively suggested that OATP1B1, OATP1B3 or OATP2B1 in monkeys could mediate roughly a similar hepatic uptake of various OATP probes. Recombinant monkey OATPs would be good experimental tools for in vitro hepatic uptake in cell systems.     

Quercetin‐3‐rhamnoglucoside (rutin) stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1

Quercetin‐3‐rhamnoglucoside (rutin) has a wide spectrum of biochemical and pharmacological activities. Rutin is absorbed mainly in its unmetabolized form. Organic anion transporting polypeptide (OATP) 2B1 is a major uptake transporter in the intestine. Thus, it is important for the prevention of adverse events to understand drug interactions mediated by OATP2B1 in the absorption process. This study assessed the effect of rutin on transport by OATP2B1. Rutin stimulated the uptake of estrone‐3‐sulfate (E‐3‐S), taurocholic acid (TCA), cholic acid (CA) and rosuvastatin by OATP2B1, but not p‐coumaric acid or ferulic acid. The EC₅₀ of rutin for transport by OATP2B1 was 2.32 μm . The K_m value of E‐3‐S for OATP2B1 in the presence of rutin (9.21 μm ) was almost the same as that in the absence of rutin (8.53 μm ). On the other hand, the V_max of E‐3‐S transport by OATP2B1 in the presence of rutin (270 pmol/mg protein/min) was 1.2‐fold higher than that in the absence of rutin (218 pmol/mg protein/min). Moreover, the expression level of OATP2B1 on the cell membrane was increased by treatment with rutin for 5 min without alteration of the total OATP2B1 expression level. Moreover, the increase in the localization of OATP2B1 at the cell surface was detected by the immunocytochemistry. The stimulatory effect of rutin is a little weak but may affect the absorption of OATP2B1 substrates, because rutin is taken daily in foods and its intestinal concentration would reach the stimulatory range of OATP2B1. Copyright © 2014 John Wiley & Sons, Ltd.     

Pre‐incubation with cyclosporine A potentiates its inhibitory effects on pitavastatin uptake mediated by recombinantly expressed cynomolgus monkey hepatic organic anion transporting polypeptide

Cyclosporine A, an inhibitor of hepatic organic anion transporting polypeptides (OATPs), reportedly increased plasma concentrations of probe substrates, although its maximum unbound blood concentrations were lower than the experimental half‐maximal inhibitory (IC₅₀) concentrations. Pre‐incubation with cyclosporine A in vitro before simultaneous incubation with probes has been reported to potentiate its inhibitory effects on recombinant human OATP‐mediated probe uptake. In the present study, the effects of cyclosporine A and rifampicin on recombinant cynomolgus monkey OATP‐mediated pitavastatin uptake were investigated in pre‐ and simultaneous incubation systems. Pre‐incubation with cyclosporine A, but not with rifampicin, decreased the apparent IC₅₀ values on recombinant cynomolgus monkey OATP1B1‐ and OATP1B3‐mediated pitavastatin uptake. Application of the co‐incubated IC₅₀ values toward R values (1 + [unbound inhibitor]_{inlet to the liver, theoretically maximum}/inhibition constant) in static models, 1.1 in monkeys and 1.3 in humans, for recombinant cynomolgus monkey and human OATP1B1‐mediated pitavastatin uptake might result in the poor prediction of drug interaction magnitudes. In contrast, the lowered IC₅₀ values after pre‐incubation with cyclosporine A provided better prediction with R values of 3.9 for monkeys and 2.7 for humans when the estimated maximum cyclosporine A concentrations at the inlet to the liver were used. These results suggest that the enhanced inhibitory potential of perpetrator medicines by pre‐incubation on cynomolgus monkey OATP‐mediated pitavastatin uptake in vitro could be of value for the precise estimation of drug interaction magnitudes in silico, in accordance with the findings from pre‐administration of inhibitors on pitavastatin pharmacokinetics validated in monkeys. Copyright © 2016 John Wiley & Sons, Ltd.     

Honey flavonoids inhibit hOATP2B1 and hOATP1A2 transporters and hOATP-mediated rosuvastatin cell uptake in vitro

1. Some flavonoids contained in the common diet have been shown to interact with important membrane uptake transporters, including organic anion transporting polypeptides (OATPs). OATP2B1 and OATP1A2 expressed in the apical membrane of human enterocytes may significantly contribute to the intestinal absorption of drugs, e.g. statins. This study is aimed at an evaluation of the inhibitory potency of selected food honey flavonoids (namely galangin, myricetin, pinocembrin, pinobanksin, chrysin and fisetin) toward hOATP2B1 and hOATP1A2 as well as at examining their effect on the cellular uptake of the known OATP substrate rosuvastatin.

2. Cell lines overexpressing the hOATP2B1 or hOATP1A2 transporter were employed as in vitro model to determine the inhibitory potency of the flavonoids toward the OATPs.

3. Chrysin, galangin and pinocembrin were found to inhibit both hOATP2B1 and hOATP1A2 in lower or comparable concentrations as the known flavonoid OATP inhibitor quercetin. Galangin, chrysin and pinocembrin effectively inhibited rosuvastatin uptake by hOATP2B1 with IC₅₀ ～1–10?μM. The inhibition of the hOATP1A2-mediated transport of rosuvastatin by these flavonoids was weaker.

4. The found data indicate that several of the tested natural compounds could potentially affect drug cellular uptake by hOATP2B1 and/or hOATP1A2 at relative low concentrations, a finding which suggests their potential for food–drug interactions.     

Characterization of ursodeoxycholic and norursodeoxycholic acid as substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and NTCP

Ursodeoxycholic acid (UDCA) is the only approved treatment for primary biliary cirrhosis, and norursodeoxycholic acid (norUDCA) is currently tested in clinical trials for future treatment of primary sclerosing cholangitis because of beneficial effects in cholestatic Mdr2 knock‐out mice. Uptake of UDCA and norUDCA into hepatocytes is believed to be a prerequisite for subsequent metabolism and therapeutic action. However, the molecular determinants of hepatocellular uptake of UDCA and norUDCA are poorly understood. We therefore investigated whether UDCA and norUDCA are substrates of the hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1 and Na⁺‐taurocholate co‐transporting polypeptide (NTCP), which are localized in the basolateral membrane of hepatocytes. Uptake of [³H]UDCA and [¹⁴C]norUDCA into Human embryonic kidney (HEK) cells stably expressing OATP1B1, OATP1B3, OATP2B1 or NTCP was investigated and compared with uptake into vector control cells. Uptake ratios were calculated by dividing uptake into transporter‐transfected cells by uptake into respective control cells. Uptake ratios of OATP1B1‐, OATP1B3‐ and OATP2B1‐mediated UDCA and norUDCA uptake were at maximum 1.23 and 1.49, respectively. Uptake of UDCA was significantly higher into HEK‐NTCP cells only at the lowest tested concentration (1 μM, p < 0.001) compared with the control cells with an uptake ratio of 1.34‐fold. NorUDCA was not significantly transported by NTCP. The low uptake rates suggest that OATP1B1, OATP1B3, OATP2B1 and NTCP are not relevant for hepatocellular uptake and effects of UDCA and norUDCA in human beings.     

Modulation of transporter activity of OATP1B1 and OATP1B3 by the major active components of Radix Ophiopogonis

Abstract

1. Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3.

2. The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D′ (OPD′) with EC₅₀ calculated to be 11.33?±?2.78 and 4.62?±?0.64?μM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD′ with EC₅₀ calculated to be 6.00?±?1.60, 13.64?±?4.07, 10.41?±?1.28 and 3.68?±?0.85?μM, respectively.

3. The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD′ with an EC₅₀ of 14.95?±?1.62?μM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD′ with an IC₅₀ of 4.44?±?1.10?μM, and barely affected by MA, MB and OPD.

4. The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.

     

Identification of natural products as modulators of OATP2B1 using LC-MS/MS to quantify OATP-mediated uptake

Context: Organic anion-transporting polypeptide 2B1 (OATP2B1) which is highly expressed in enterocytes and hepatocytes could be a key determinant for the intestinal absorption and hepatic uptake of its substrate drugs. Natural products are commonly used in traditional Chinese medicine, foods, and beverages.

Objective: The objective of this study is to determine the OATP2B1-mediated drug interactions that could occur between natural products and OATP2B1 substrate drugs.

Materials and methods: Human OATP2B1 was transiently expressed in human embryonic kidney (HEK293) cells and characterized by immunofluorescence, Western blot, and uptake assay. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods for detecting OATP2B1 substrates estrone-3-sulfate (E3S) and three statins had been developed and were employed to investigate the effects of 27 frequently used natural products on the function of OATP2B1. Uptake of 5?μM E3S and 1?μM statins in the absence or presence of natural products was measured at 37?°C for 2?min with empty vector- and OATP2B1-transfected HEK293 cells. The IC₅₀ values of inhibitors for OATP2B1-mediated 5?μM E3S uptake were determined.

Results: Our results showed that mulberrin, scutellarin, quercetin, and glycyrrhetinic acid were strong inhibitors of OATP2B1-mediate E3S uptake with IC₅₀ values being 1.8, 2.0, 7.5, and 13.0?μM, which were comparable with their plasma concentrations in clinical trials. They also inhibited OATP-mediated uptake of atorvastatin, fluvastatin, and rosuvastatin. These results indicated that clinically relevant drug interactions could occur between these natural compounds and OATP2B1 substrate drugs.

Discussion and conclusion: The information obtained from this study might be helpful to predict and to avoid potential OATP2B1-mediated drug interactions.     

Interaction of deoxyschizandrin and schizandrin B with liver uptake transporters OATP1B1 and OATP1B3

1. Deoxyschizandrin and schizandrin B have diverse pharmacological effects, including hepatoprotective activity. We aim to study their hepatic uptake and their effects on the hepatic uptake of other clinical drugs mediated by OATP1B1 and OATP1B3.

2. Deoxyschizandrin exhibited a high affinity for OATP1B1 with K_m of 17.61?±?0.43?μM but a low affinity for OATP1B3. Similarly, schizandrin B also showed a strong affinity for OATP1B1 with K_m of 18.45?±?1.23?μM but a weak affinity for OATP1B3.

3. Atorvastatin and rifampicin could inhibit the uptake of deoxyschizandrin and schizandrin B mediated by OATP1B1.

4. Intriguingly, both deoxyschizandrin and schizandrin B significantly promoted the uptake of atorvastatin (with EC₅₀ of 50.58?±?8.08 and 24.70?±?5.82 µM, respectively) and rosuvastatin (with EC₅₀ of 13.46?±?2.70 and 8.99?±?4.73 µM, respectively) mediated by OATP1B1. Deoxyschizandrin could markedly promote the uptake of fluvastatin but inhibit the uptake of sodium taurocholate (TCNa) mediated by OATP1B1.

5. The promotion on hepatic uptake of statins mediated by OATP1B1 might lead to enhanced efficacy of cholesterol lowering and reduced risk of myopathy for hyperlipidemia patients when given statins together with deoxyschizandrin or schizandrin B.     

Major Active Components in Grapefruit,Orange, and Apple Juices Responsible for OATP2B1-Mediated Drug Interactions

We aimed to explore the major active components in grapefruit juice (GFJ), orange juice (OJ), and apple juice (AJ) that are responsible for OATP2B1‐mediated drug interactions, by means of in vitro studies using Xenopus oocytes expressing OATP2B1 with a typical OATP2B1 substrate, estrone‐3‐sulfate. All three juices inhibited OATP2B1‐mediated estrone‐3‐sulfate uptake with half‐maximum inhibition (IC₅₀) values of 0.222% (GFJ), 0.807% (OJ), and 2.27% (AJ). Eight major flavonoids (naringin, naringenin, hesperidin, hesperetin, phloridzin, phloretin, quercetin, and kaempferol) contained in the juices inhibited OATP2B1‐mediated estrone‐3‐sulfate uptake with IC₅₀ values of 4.63, 49.2, 1.92, 67.6, 23.2, 1.31, 9.47, and 21.3 µM, respectively. When the concentration–IC₅₀ ratios ([C]/IC₅₀) of these flavonoids in GFJ, OJ, and AJ were calculated, values of [C]/IC₅₀ ≥ 100 were obtained for naringin in GFJ and hesperidin in OJ. No flavonoid in AJ showed a ratio higher than unity. However, significant inhibition of OATP2B1 was observed with a mixture of phloridzin, phloretin, hesperidin, and quercetin at the concentrations present in AJ. In conclusion, our results indicate that naringin and hesperidin are the major OATP2B1 inhibitors in GFJ and OJ, respectively, whereas a combination of multiple components appears to be responsible for OATP2B1 inhibition by AJ.     

Impact of curcumin on the pharmacokinetics of rosuvastatin in rats and dogs based on the conjugated metabolites

1. Plasma concentrations of curcumin-O-glucuronide (COG) and curcumin-O-sulfate (COS) significantly increased after Sprague-Dawley rats dealt with the Oatp inhibitor rifampicin, with the C_max ascending 2.9 and 6.7 times, and the AUC_0–∞ ascending 4.4 and 10.8 times, respectively. When pretreated with the Oat inhibitor probenecid, the C_max increased 4.4 and 20 times, and the AUC_0–∞ increased 3.2 and 13.9 times, respectively. The results suggested that COG and COS may be the substrates of Oatp and Oat.

2. The accumulation of curcumin significantly increased in organic anion transporting polypeptide (OATP)- and organic anion transporter (OAT)-transfected human embryonic kidney (HEK) 293 systems, which suggested that curcumin was a substrate of OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT3; and COG was a substrate of OATP1B1, OATP1B3, and OAT3.

3. Inhibition study using rosuvastatin as the substrate in OATP1B1- and OATP1B3-transfected cells indicated that curcumin was an OATP1B1 and 1B3 inhibitor, with IC₅₀ at 5.19?±?0.05 and 3.68?±?0.05?μM, respectively; the data for COG were 1.04?±?0.01 and 1.08?±?0.02?μM, respectively. COS was speculated to be an inhibitor of hepatic OATP1B1 as calculated using the ADMET Predictor.

4. COG and COS are substrates and inhibitors of OATP/Oatp. Co-administration of curcumin significantly increased rosuvastatin concentration in rat and dog plasma.     

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by K_m values of 22.5 and 14.1 µM, and V_max values of 98.3 and 5.8 pmol/(million cells ? min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter‐mediated uptake of NaFluo was observed in OATP1B1‐ and OATP1B3‐transfected cells (K_m = 4.2 and 10.9 µM; V_max = 30.9 and 135 [pmol/(mg protein ? min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B‐mediated drug interactions in fluorescence‐based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5018–5030, 2011     

Transmembrane transport of steviol glucuronide and its potential interaction with selected drugs and natural compounds

Steviol glucuronide (SVG) is the major metabolite derived from steviol, the aglycone of stevioside and rebaudioside A. After the ingestion of stevioside and rebaudioside A, SVG is formed and excreted into the urine in humans. In the present study, transporter mediated efflux and uptake of SVG was investigated in order to understand molecular mechanisms underlying its renal clearance. Results showed that SVG was not a substrate of efflux transporters BCRP, MRP2, MATE1 or P-gp. In contrast, OAT3 played a predominant role in the uptake of SVG in comparison to OATP1B1, OATP1B3, or OATP2B1. Quercetin, telmisartan, diclofenac, and mulberrin displayed a relatively strong inhibition against OAT3 mediated uptake of SVG with IC₅₀ values of 1.8, 2.9, 8.0, and 10.0 μM, respectively. Because OAT3 is a major uptake transporter in the kidney, inhibition of OAT3 activity may alter SVG's renal clearance by drugs and natural compounds that are used concomitantly with stevia leaf extracts.     

Alteration of the intravenous and oral pharmacokinetics of valsartan via the concurrent use of gemfibrozil in rats

The present study aimed to examine the potential pharmacokinetic drug interaction between valsartan and gemfibrozil. Compared with the control given valsartan (10 mg/kg) alone, the concurrent use of gemfibrozil (10 mg/kg) significantly (p < 0.05) increased the oral exposure of valsartan in rats. In the presence of gemfibrozil, the C_max and AUC of oral valsartan increased by 1.7‐ and 2.5‐fold, respectively. Consequently, the oral bioavailability of valsartan was significantly higher (p < 0.05) in the presence of gemfibrozil compared with that of the control group. Furthermore, the intravenous pharmacokinetics of valsartan (1 mg/kg) was also altered by pretreatment with oral gemfibrozil (10 mg/kg). The plasma clearance of valsartan was decreased by two‐fold in the presence of gemfibrozil, while the plasma half‐life was not altered. In contrast, both the oral and intravenous pharmacokinetics of gemfibrozil were not affected by the concurrent use of valsartan. The cellular uptake of valsartan and gemfibrozil was also investigated by using cells overexpressing OATP1B1 or OATP1B3. Gemfibrozil and gemfibrozil 1‐O‐β glucuronide inhibited the cellular uptake of valsartan with IC₅₀ values (µm ) of 39.3 and 20.4, respectively, in MDCK/OATP1B1, while they were less interactive with OATP1B3. The cellular uptake of gemfibrozil was not affected by co‐incubation with valsartan in both cells. Taken together, the present study suggests the potential drug interaction between valsartan and gemfibrozil, at least in part, via the OATP1B1‐mediated transport pathways during hepatic uptake. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.     

PBPK modeling of irbesartan: incorporation of hepatic uptake

Physiological based pharmacokinetic (PBPK) modeling is now commonly used in drug development to integrate human or animal physiological data in order to predict pharmacokinetic profiles. The aim of this work was to construct and refine a PBPK model of irbesartan taking into account its active uptake via OATP1B1/B3 in order to predict more accurately its pharmacokinetic profile using Simcyp^®. The activity and expression of the human hepatocyte transporters OATP1B1 and OATP1B3 were studied. The relative activity factors (RAFs) for OATP1B1 and OATP1B3 transporters were calculated from intrinsic clearances obtained by concentration dependent uptake experiments in human hepatocytes and HEK overexpressing cells: RAF_1B1 using estrone‐3‐sulfate and pitavastatine clearances, and RAF_1B3 using cholecystokinine octapeptide (CCK‐8) clearances. The relative expression factor (REF) was calculated by comparing immunoblotting of hepatocytes (REF_HH) or tissues (REF_tissue) with those of overexpressing HEK cells for each transporter. These scaling factors were applied in a PBPK model of irbesartan using the Simcyp® simulator. Pharmacokinetic simulation using REF_HH (1.82 for OATP1B1, 8.03 for OATP1B3) as an extrapolation factor was the closest to the human clinical pharmacokinetic profile of irbesartan. These investigations show the importance of integrating the contribution of the active uptake of a drug in the liver to improve PBPK modeling. Copyright © 2015 John Wiley & Sons, Ltd.     

Interactions of crizotinib and gefitinib with organic anion-transporting polypeptides (OATP)1B1, OATP1B3 and OATP2B1: gefitinib shows contradictory interaction with OATP1B3

1.?The drug–drug interaction (DDI) mediated by organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and OATP2B1 has a major impact on the hepatic clearance of drugs. The effects of tyrosine kinase inhibitors (TKIs) on OATPs have not been well studied. In the present study, we evaluated the contribution of OATPs to the hepatic uptake of crizotinib and gefitinib and the interaction of those TKIs with OATPs to estimate DDIs.

2.?To clarify whether crizotinib and gefitinib were substrates for OATPs, we performed uptake studies. We examined the effects of the TKIs on uptake of typical substrates and fluvastatin via OATPs. IC₅₀ and EC₅₀ values of the TKIs were calculated.

3.?OATP1B3- and OATP2B1-mediated crizotinib uptake and OATP2B1-mediated gefitinib uptake were observed. Gefitinib accelerated OATP1B3-mediated [³H]TCA uptake and inhibited OATP2B1-mediated [³H]E₃S uptake. On the other hand, gefitinib inhibited OATP1B1- and OATP2B1-mediated fluvastatin uptake.

4.?We provided basic information to estimate the DDI on OATPs caused by TKIs. The DDI on OATPs caused by gefitinib could occur in a normal clinical situation. And the uptake of crizotinib into the intrahepatocellular environment via OATPs may induce DDI and liver damage. We therefore emphasize the necessity of careful use of TKIs.     

Long-Lasting Inhibitory Effects of Saquinavir and Ritonavir on OATP1B1-Mediated Uptake

Previously, we reported a long-lasting inhibition of transport mediated by organic anion-transporting polypeptides (OATPs) in humans and rats by cyclosporin A (CsA). In the present study, we examined the effects of several other compounds on OATP1B1-mediated transport, with a focus on long-lasting inhibition. Effects of coincubation, preincubation, or preincubation plus coincubation of 12 compounds on uptake of estrone 3-sulfate (E₁S) in OATP1B1-expressing HEK293T cells were examined. The OATP1B1 inhibitors used in the present study inhibited OATP1B1-mediated uptake of E₁S in a concentration-dependent manner. Among them, saquinavir and ritonavir in addition to CsA exhibited long-lasting inhibitory effects on OATP1B1-mediated transport of E₁S at ≥5 and 25 μM, respectively, even after they were washed out from the incubation buffer. After preincubation with saquinavir, its inhibitory effect on OATP1B1 remained for at least 6 h, whereas the effect of ritonavir did not remain. Protein expression of OATP1B1 was not altered by preincubation with 25 μM saquinavir or ritonavir. The present study firstly showed that saquinavir and ritonavir as well as CsA have long-lasting inhibitory effects on OATP1B1. But, at plasma unbound concentrations of saquinavir and ritonavir in clinical situations, they may not cause long-lasting inhibition of OATP1B1.     

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.     

Characterization of Hepatobiliary Transport Systems of a Novel α4β1/α4β7 Dual Antagonist, TR-14035

Purpose Our previous pharmacokinetic studies have demonstrated that TR-14035, a novel dual antagonist for α4β1/α4β7 integrin, selectively and strongly accumulated in the liver and was mainly excreted in bile as an unchanged drug. In the present study, we investigated the hepatobiliary transport system in detail.Materials and Methods Uptake by hepatocytes and organic anion transporting polypeptide (OATP)-expressing Xenopus laevis oocytes or Flp-In-293 cells was performed in vitro. Biliary excretion was investigated in mdr1a/b-knockout mice, Bcrp-knockout mice and Mrp2-defective Eisai hyperbilirubinemic rats (EHBRs).Results TR-14035 was taken up by rat and human hepatocytes by an apparently single saturable mechanism with K _m of 6.7 and 2.1 μM, respectively, and taurocholate and digoxin reduced this uptake. OATP1B1/OATP-C and OATP1B3/OATP8 expressed in oocytes mediated the TR-14035 uptake with K _m of 7.5 and 5.3 μM, respectively. OATP1B1*15, a genetic variant of OATP1B1, exhibited a decreased transport of TR-14035 compared with OATP1B1*1a. Biliary excretion and total body clearance of unchanged TR-14035 in EHBRs were significantly lower than those in normal rats, while there was no difference in the clearances between wild and mdr1a/b- or Bcrp-knockout mice.Conclusion These results indicate that OATP1B1 and OATP1B3 are at least partly responsible for the accumulation of TR-14035 into hepatocytes, and Mrp2 principally mediates the biliary excretion of TR-14035. Furthermore, genetic polymorphisms of OATP1B1 may cause an interindividual variability in the pharmacokinetics of TR-14035.