Intestinal absorption of HMG-CoA reductase inhibitor pitavastatin mediated by organic anion transporting polypeptide and P-glycoprotein/multidrug resistance 1

The purpose of this study was to investigate the involvement of organic anion transporting polypeptide (OATP/Oatp) and P-glycoprotein (P-gp)/multidrug resistance 1 (MDR1/Mdr1) in intestinal absorption of pitavastatin. Pitavastatin was found to be a substrate for human OATP1A2, OATP2B1, and MDR1 and rat Oatp1a5, Oatp2b1, and Mdr1a in experiments using transporter-expressing Xenopus oocytes and LLC-PK1 cell systems. Naringin inhibited Oatp1a5- and Mdr1a-mediated transport of pitavastatin with IC(50) values of 18.5 and 541 μM, respectively. The difference in the IC(50) values of naringin for Oatp1a5 and Mdr1a-mediated pitavastatin transport may account for the complex concentration-dependent effect of naringin on the intestinal absorption of pitavastatin. Rat intestinal permeability of pitavastatin measured by the in situ closed-loop perfusion method was indeed significantly reduced by 200 μM naringin, but was significantly increased by 1000 μM naringin. Furthermore, the permeability was significantly increased by elacridar, a potent inhibitor of Mdr1, while the permeability was significantly decreased in the presence of both elacridar and naringin, suggesting that Oatp1a5 and Mdr1a are both involved in intestinal absorption of pitavastatin. Our present results indicate that OATP/Oatp and MDR1/Mdr1 play roles in the intestinal absorption of pitavastatin as influx and efflux transporters, respectively.     

Active intestinal absorption of fluoroquinolone antibacterial agent ciprofloxacin by organic anion transporting polypeptide, Oatp1a5

Fluoroquinolone antimicrobial drugs are absorbed efficiently after oral administration despite of their hydrophilic nature, implying an involvement of carrier‐mediated transport in their membrane transport process. It has been that several fluoroquinolones are substrates of organic anion transporter polypeptides OATP1A2 expressed in human intestine derived Caco‐2 cells. In the present study, to clarify the involvement of OATP in intestinal absorption of ciprofloxacin, the contribution of Oatp1a5, which is expressed at the apical membranes of rat enterocytes, to intestinal absorption of ciprofloxacin was investigated in rats. The intestinal membrane permeability of ciprofloxacin was measured by in situ and the vascular perfused closed loop methods. The disappeared and absorbed amount of ciprofloxacin from the intestinal lumen were increased markedly in the presence of 7,8‐benzoflavone, a breast cancer resistance protein inhibitor, and ivermectin, a P‐glycoprotein inhibitor, while it was decreased significantly in the presence of these inhibitors in combination with naringin, an Oatp1a5 inhibitor. Furthermore, the Oatp1a5‐mediated uptake of ciprofloxacin was saturable with a K_m value of 140 µm , and naringin inhibited the uptake with an IC₅₀ value of 18 µm by Xenopus oocytes expressing Oatp1a5. Naringin reduced the permeation of ciprofloxacin from the mucosal‐to‐serosal side, with an IC₅₀ value of 7.5 µm by the Ussing‐type chamber method. The estimated IC₅₀ values were comparable to that of Oatp1a5. These data suggest that Oatp1a5 is partially responsible for the intestinal absorption of ciprofloxacin. In conclusion, the intestinal absorption of ciprofloxacin could be affected by influx transporters such as Oatp1a5 as well as the efflux transporters such as P‐gp and Bcrp. Copyright © 2012 John Wiley & Sons, Ltd.     

Loss of organic anion transporting polypeptide 1a1 increases deoxycholic acid absorption in mice by increasing intestinal permeability

Deoxycholic acid (DCA) is a known hepatotoxicant, a tissue tumor promoter, and has been implicated in colorectal cancer. Male mice are more susceptible to DCA toxicity than female mice. Organic anion transporting polypeptide 1a1 (Oatp1a1), which is known to transport bile acids (BAs) in vitro, is predominantly expressed in livers of male mice. In addition, the concentrations of DCA and its taurine conjugate (TDCA) are increased in serum of Oatp1a1-null mice. To investigate whether Oatp1a1 contributes to the gender difference in DCA toxicity in mice, wild-type (WT) and Oatp1a1-null mice were fed a 0.3% DCA diet for 7 days. After feeding DCA, Oatp1a1-null mice had 30-fold higher concentrations of DCA in both serum and livers than WT mice. Feeding DCA caused more hepatotoxcity in Oatp1a1-null mice than WT mice. After feeding DCA, Oatp1a1-null mice expressed higher BA efflux-transporters (bile salt-export pump, organic solute transporter (Ost)α/β, and multidrug resistance-associated protein [Mrp]2) and lower BA-synthetic enzymes (cytochrome P450 [Cyp]7a1, 8b1, 27a1, and 7b1) in livers than WT mice. Intravenous administration of DCA and TDCA showed that lack of Oatp1a1 does not decrease the plasma elimination of DCA or TDCA. After feeding DCA, the concentrations of DCA in ileum and colon tissues are higher in Oatp1a1-null than in WT mice. In addition, Oatp1a1-null mice have enhanced intestinal permeability. Taken together, the current data suggest that Oatp1a1 does not mediate the hepatic uptake of DCA or TDCA, but lack of Oatp1a1 increases intestinal permeability and thus enhances the absorption of DCA in mice.     

Functional pleiotropy of organic anion transporting polypeptide OATP2B1 due to multiple binding sites

The purpose of this study was to examine whether the presence of multiple binding sites can explain the pleiotropy of substrate recognition by OATP2B1, using Xenopus oocytes expressing OATP2B1. OATP2B1-mediated uptake of estrone-3-sulfate apparently exhibited biphasic saturation kinetics, with Km values of 0.10 ± 0.05 and 29.9 ± 12.1 μM and Vmax values of 14.1 ± 6.4 and 995 ± 273 fmol/min/oocyte for high- and low-affinity sites, respectively. Contribution analysis revealed that transport of estrone-3-sulfate mediated by high- and low-affinity sites on OATP2B1 could be evaluated at the concentrations of 0.005 and 50 μM, respectively. pH-dependence study of OATP2B1-mediated estrone-3-sulfate uptake suggested that high- and low-affinity sites show different pH sensitivity. When the inhibitory effect of 12 compounds on estrone-3-sulfate uptake by high- and low-affinity sites on OATP2B1 was examined, 4 compounds appeared to be inhibitors of the high-affinity site on OATP2B1. Two other compounds appeared to be inhibitors for the low-affinity site and four others were inhibitory at both sites. These results indicated the presence of multiple binding sites on OATP2B1 with different affinity for drugs. Accordingly, it is likely that drug-drug and drug-beverage interactions occur only when two drugs share the same binding site on OATP2B1.     

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.     

Stimulatory effect on the transport mediated by organic anion transporting polypeptide 2B1

Drug-drug interaction(DDI)is one of causes of adverse drug events and can result in lifethreatening consequences.Organic anion-transporting polypeptide(OATP)2B1 is a major uptake transporter in the intestine and contributes to transport various clinically used therapeutic agents.The intestine has a high risk of DDI,because it has a special propensity to be exposed to a high concentration of drugs.Thus,understanding drug interaction mediated by OATP2B1 in the absorption process is important for the prevention of adverse drug events,including decrease in the therapeutic effect of co-administered drugs.Acute drug interaction occurs through the direct inhibitory effect on transporters,including OATP2B1.Moreover,some compounds such as clinically used drugs and food components have an acute stimulatory effect on transport of co-administered drugs by OATP2B1.This review summarizes the acute stimulatory effect on the transport mediated by OATP2B1 and discusses the mechanisms of the acute stimulatory effects of compounds.There are two types of acute stimulatory effects,substrate-independent and-dependent interactions on OATP2B1 function.The facilitating translocation of OATP2B1 to the plasma membrane is one of causes for the substrate-independent acute stimulatory effect.On the contrary,the substrate-dependent effect is based on the direct binding to the substrate-binding site or allosteric progesterone-binding site of OATP2B1.     

Effects of grapefruit juice on intestinal P-glycoprotein: evaluation using digoxin in humans

STUDY OBJECTIVES: To determine the effects of grapefruit juice on the pharmacokinetics of oral digoxin, a P-glycoprotein substrate not metabolized by cytochrome P450 3A4, in healthy volunteers, and to assess whether polymorphic multidrug-resistance-1 (MDR1) expression contributes to interindividual variability in digoxin disposition. DESIGN: Prospective, open-label, unblinded, crossover study. SETTING: University research center. SUBJECTS: Seven healthy adult volunteers (four men, three women). INTERVENTION: Each subject received a single oral dose of digoxin 1.0 mg with water or grapefruit juice with at least a 2-week washout between treatments. During the grapefruit juice phase, juice was administered 3 times/day for 5 days before digoxin administration to maximize any effect on P-glycoprotein. MEASUREMENTS AND MAIN RESULTS: Digoxin pharmacokinetics in the presence and absence of grapefruit juice were compared. The MDR1 exon 26 C3435T genotype was determined by real-time polymerase chain reaction. Compared with water, grapefruit juice significantly reduced the digoxin absorption rate constant (3.0 +/- 2.4 to 1.2 +/- 1.0 hr(-1), p<0.05) and increased absorption lag time (0.32 +/- 0.12 to 0.53 +/- 0.34 hr, p<0.05). Grapefruit juice did not affect digoxin maximum concentration (Cmax), area under the curve (AUC), elimination half-life, or renal clearance. The effect of grapefruit juice on digoxin Cmax (-45% to +41%) and AUC(0-4) (-29% to +25%) varied substantially among subjects and was inversely correlated with the values during the water phase. Trends toward higher digoxin Cmax AUC, and absorption rate constant during the water phase were found in CC homozygotes compared with subjects carrying a T allele. CONCLUSION: Inhibition of intestinal P-glycoprotein does not appear to play an important role in drug interactions involving grapefruit juice. Interindividual variability in response to grapefruit juice may be related to the balance of intestinal drug uptake and efflux transport.     

Insulin stimulates transport of organic anion compounds mediated by organic anion transporting polypeptide 2B1 in the human intestinal cell line Caco-2

Organic anion transporting polypeptide 2B1 (OATP2B1) is the major uptake transporter in the intestine, and transports various clinically used therapeutic agents. Insulin acts through the insulin receptor in targeted cells, and Rab8A is one of the insulin signaling pathways. The small intestine in humans also expresses insulin receptor and Rab8A. It has been reported that insulin stimulates peptide transporter 1 (PEPT1) expression at the apical membrane and increases uptake of PEPT1 substrates in small intestine epithelial model cells (Caco-2 cells). However, the effect of insulin on OATP2B1 in the small intestine has not been fully investigated. We found that Rab8A was associated with OATP2B1-mediated estrone-3-sulfate (E3S) uptake. Insulin stimulated the uptake of E3S by Caco-2 cells and the enhancement was sustained for 120 min. The Vmax value of E3S uptake significantly increased upon insulin exposure. Caco-2 cells treated with insulin showed increased OATP2B1 expression at the cell surface. The apical-to-basal transport of E3S was also increased by insulin. The increase of E3S transport was inhibited by the cold condition (4 °C) or the OATP2B1 inhibitor, taurocholate. These results indicate that insulin acts on the small intestine to increase OATP2B1-mediated absorption.     

有机阴离子转运多肽1A2基因多态性的研究进展

目的为有机阴离子转运多肽1A2(organic anion transporting polypeptide1A2,OATP1A2)基因多态性的深入研究及临床用药提供参考。方法查阅了国内外相关文献25篇,对其进行了分析和总结。结果 OATP1A2具有明显的基因多态性,516A>C、404A>T等单体型可显著影响OATP1A2的转运活性,-1 105G>A、-1 032G>A等单体型可明显影响药物体内药动学过程,从而导致临床用药的个体差异。结论目前有关OATP1A2基因多态性的研究多基于体外实验研究其不同多态变体转运能力的差异,更多进一步的OATP1A2基因多态性临床相关性研究具有重要意义。     

Role of organic anion transporting polypeptide 2 in pharmacokinetics of digoxin and beta-methyldigoxin in rats

Recently, we found that potent P-glycoprotein (P-gp) inhibitors, such as verapamil and cyclosporin A, markedly modulated the pharmacokinetics of digoxin in rats, whereas they did not affect beta-methyldigoxin pharmacokinetics significantly. Digoxin is also a substrate of rat organic anion transporting polypeptide 2 (Oatp2). Here, we compared the magnitude of Oatp2-mediated drug interaction of digoxin and beta-methyldigoxin using amiodarone as an Oatp2 inhibitor in rats. Amiodarone (20 mg/kg) given intravenously significantly increased plasma levels and decreased biliary excretion, liver distribution, and intestinal distribution of digoxin administered intravenously at a dose of 10 mug/kg. Amiodarone also significantly decreased biliary excretion and liver distribution of beta-methyldigoxin, but the change in plasma levels of beta-methyldigoxin was quite small. These findings may give a clue in selecting these cardiac glycosides in clinical pharmacotherapy for patients receiving multiple drugs towards escape from Oatp2-mediated drug interactions.     

Effects of natural products on the function of human organic anion transporting polypeptide 1B1

In this study, the effects of 136 naturally occurring products, which have been reported to play important roles in modification of Cytochrome P450 (CYP450) activities, on the uptake of estrone-3-sulfate (E3S), a typical OATP1B1 substrate, were evaluated using human embryonic kidney 293 cells stably expressing OATP1B1. At a concentration of 100 μM, 42 natural products inhibited OATP1B1-mediated [(3)H]E3S uptake by more than 50%, and five of them significantly inhibited OATP1B1-mediated [(3)H]E3S by more than 80% with the following rank order of potency: quercetin > astragaloside IV > icariin > glycyrrhizic acid > ginsenoside Rc. Inhibitory effects of these natural products on OATP1B1 activity were in a concentration-dependent manner. 11 natural compounds were found exhibiting greater than 50% inhibition at 30 μM with IC(50) values ranging from 14.6 ± 3.3 to 28.5 ± 3.0 μM. In conclusion, our data suggest that modification of OATP1B1 transport activity by these natural occurring products may be a mechanism for natural product-drug interactions in humans.     

Substrate-dependent effects of molecular-targeted anticancer agents on activity of organic anion transporting polypeptide 1B1

1. Organic anion-transporting polypeptide 1B1 (OATP1B1) plays an important role in the hepatic uptake of a broad range of substrate drugs. In vitro experiments show that molecular-targeted agents do not always have similar effects on OATP1B1 activity.

2. The purpose of this study was to clarify whether the effects of molecular-targeted agents on OATP1B1 are substrate-dependent. We used OATP1B1-transfected cells to compare the effects of molecular-targeted agents on OATP1B1-mediated uptake of fluorescein (FL), 2′,7′-dichlorofluorescein (DCF), atorvastatin, SN-38 and valsartan.

3. Cabozantinib, cediranib, neratinib, pazopanib, regorafenib, sorafenib and tivantinib did not affect or only slightly affected OATP1B1-mediated substrate uptake. Nilotinib and lenvatinib moderately and strongly inhibited OATP1B1-mediated substrate uptake, respectively. In contrast, afatinib stimulated OATP1B1-mediated uptake of FL and SN-38, ceritinib stimulated that of valsartan, and nintedanib stimulated that of FL and valsartan. In addition, the effects of afatinib, ceritinib and nintedanib on OATP1B1 activity differed markedly depending on the type of substrate. Afatinib, ceritinib and nintedanib had a substrate-dependent effect on OATP1B1 activity.

4. We conclude that the evaluation of OATP1B1 activity using only a single probe substrate for some molecular-targeted agents may lead to a faulty understanding of their mechanisms of drug interactions.     

Bioactive xanthones with effect on P-glycoprotein and prediction of intestinal absorption

Our research group has been focusing in the discovery of potential antitumor small molecules based on the xanthone scaffold. However, a serious obstacle in the field of cancer therapy is the multidrug resistance (MDR) phenotype, most often caused by the overexpression of P-glycoprotein (P-gp). Another limitation to development of such drug candidates is the reduced information available about the bioavailability of these compounds. We have previously identified four interesting compounds as inhibitors of tumor cell growth namely two dihydroxyxanthones, a xanthonolignoid and a pyranoxanthone. Based on these considerations, it was our aim to: (i) investigate their effect on the P-gp activity; and (ii) estimate their intestinal absorption using Caco-2 cell monolayers as an intestinal model. An HPLC analysis from the in vitro permeation assay with Caco-2 cells monolayer was performed to predict the intestinal permeability of xanthonic derivatives. A rhodamine (Rh123) accumulation assay using P-gp overexpressing leukemia cells, K562Dox, incubated with the four xanthonic derivatives, was performed to investigate their P-gp inhibitory activity. A luminescence-based ATPase assay was performed to differentiate between competitive and noncompetitive P-gp inhibitors. The xanthonolignoid and the pyranoxanthone were found to increase the accumulation of Rh123 in K562Dox cell line, and both were acting by a noncompetitive P-gp inhibitory mechanism. Transport of the four xanthones occurred in the absorptive direction (P_app, 0.012–2.8 nm/s). The behavior of the xanthonolignoid and the pyranoxanthone as P-gp inhibitors and their high apparent permeability coefficients make them promising hit compounds to pursue with further studies.     

Exposure-dependent inhibition of intestinal and hepatic CYP3A4 in vivo by grapefruit juice

Consumption of typical quantities of grapefruit juice (GFJ) increases the oral bioavailability of several CYP3A4 substrates without affecting their elimination, consistent with selective inhibition of intestinal but not hepatic CYP3A4. However, increases in the AUCs of CYP3A4 substrates recently associated with the consumption of large amounts of GFJ were similar to those observed with potent inhibitors of hepatic CYP3A4. The current study compared the effects of consuming large quantities and more typical amounts of GFJ on the activity of hepatic and intestinal cytochrome P450 3A4 in vivo, employing the erythromycin breath test (EBT) and oral midazolam pharmacokinetics. This was a two-phase, randomized, placebo-controlled crossover study, with each phase conducted with a separate panel of subjects. In Phase I, 8 male volunteers were randomized to the order of receiving one glass (240 mL) of water (placebo) or double-strength (DS) GFJ tid for 2 days and then 90, 60, and 30 minutes prior to administration of probe drugs on the 3rd day. In Phase II, 16 male volunteers were randomized to the order of receiving one glass of (1) single-strength (SS) GFJ, (2) DS GFJ, and (3) water (placebo). All treatments were administered in a fasted state. There was at least a 7-day washout period between treatments. Probe drugs, administered 30 minutes or 1 hour following each treatment in Phase I or II, respectively, consisted of oral midazolam (2 mg) coadministered with IV [14G N-methyl] erythromycin (0.03 mg). The EBT was performed 20 minutes following erythromycin administration. Blood was collected during the 24 hours following probe drug administration for the analysis of midazolam pharmacokinetics. In Phase I, consumption of one glass of DS GFJ tid for 3 days increased the Cmax of midazolam 3-fold, the AUC 6-fold, and the t1/2 2-fold and decreased the amount of exhaled 14CO2 in all 8 subjects, with a mean decrease in EBT of 18%. In Phase II, consumption of one glass of DS GFJ significantly increased the AUC and Cmax of midazolam approximately 2-fold without a significant effect on the t1/2 of midazolam or the EBT. The effects of consuming one glass of SS GFJ on midazolam pharmacokinetics and the EBT were not significantly different from those of one glass of DS GFJ. It was concluded that consumption of one glass of DS GFJ tid for 3 days significantly increased the AUC, Cmax, and t1/2 of midazolam and reduced EBT values, reflecting inhibition of both hepatic and intestinal CYP3A4. In contrast, consumption of one glass of SS or DS GFJ increased midazolam AUC and Cmax, with little effect on the midazolam t1/2 and EBT values, reflecting preferential inhibition of intestinal CYP3A4. Alterations of midazolam AUC and Cmax induced by nine glasses of DS GFJ were significantly greater than those produced by one glass of SS or DS GFJ. These data suggest that GFJ inhibits intestinal and hepatic CYP3A4 in an exposure-dependent fashion and that patients taking medications that are CYP3A4 substrates are at risk for developing drug-related adverse events if they consume large amounts of grapefruit juice.     

Effect of organic anion transporting polypeptide 1B1 inhibition on the pharmacokinetics of rosuvastatin via HNF1a by ursodeoxycholic acid in vivo

AIM： The inhibition of organic anion transporting polypeptide 1B1 （OATP1B1） on the pharmacokinetics of rosuvastatin is unknown. Hence, the effect of ursodeoxycholic acid （UDCA） on the kinetics of rosuvastatin in healthy volunteers is to be investigated. METHODS： The inhibition effect of long term use of UDCA on rosuvastatin kinetics was studied in 12 subjects in a randomized, crossover study. Each subject received 500 mg UDCA once daily continuously for 14 days. A single oral dose of 20 mg rosuvastatin was given on study day 15 and 34 separated by 2 weeks. Plasma concentrations of rosuvastatin were above the limits of quantitation for up to 72 h after dosing. RESULTS： UDCA significantly increased AUC0-72 and AUC0-∞ of rosuvastatin to 146% ± 55% （P = 0.008） and 167% ± 73% （ P = 0.004） compared with those of the control group and CL/F decreased 75% ± 19% （P = 0. 003）. The results confirmed the in vitro study that UDCA inhibited OATP1B1 activity via hepatic nuclear factor 1a （HNF1a）.[第一段]     

Uptake of enalapril and expression of organic anion transporting polypeptide 1 in zonal, isolated rat hepatocytes.

Sinusoidal entry is the first obligatory process preceding intracellular drug removal in liver. Transport of the angiotensin converting enzyme inhibitor enalapril (1-750 microM with [(3)H]enalapril), a substrate of Oatp1, the sodium-independent organic anion transporting polypeptide 1 cloned from rat liver, was studied in rat hepatocytes isolated from all zones of the liver (homogeneous) and from enriched periportal (PP) and perivenous (PV) hepatocytes prepared by collagenase perfusion and zone-selective destruction with digitonin, respectively. Uptake was linear over 1 min and was concentration-dependent. Transport by the homogeneous hepatocytes (in the presence and absence of Na(+)) and PP and PV cells was described by single saturable components of similar kinetic constants (K(m) values of 344-461 microM and V(max) values of 9.5-11.6 nmol/min/10(6) cells; P >.05, ANOVA). The K(m) value for enalapril uptake in hepatocytes was of the same order of magnitude compared with that for Oatp1 expressed in HeLa cells transfected with cDNA-Oatp1 and Western blot analysis revealed similar levels of immunoreactive Oatp1 expression in PP and PV hepatocytes. However, enalapril was not taken up by Oatp2 nor by the human OATP expressed in recombinant vaccinia systems.     

Functional and structural relevance of conserved positively charged lysine residues in organic anion transporting polypeptide 1B3

The human organic anion transporting polypeptide 1B3 (OATP1B3), located in the basolateral membrane of hepatocytes, mediates the uptake of endogenous substrates such as taurocholate and drugs from blood into hepatocytes. The transport activity of OATP1B3 is influenced by positively charged amino acids, which are facing the central pore. Molecular modeling was performed to select conserved positively charged amino acids, which may influence transport activity and anchoring of OATP1B3 in the plasma membrane. The modeling revealed that Lys361 faces the pore, and Lys399 is oriented to the plasma membrane. Therefore, the mutants L361>A, L361>R, L399>A, and L399>R were generated using site-directed mutagenesis to investigate the impact of the positive charges on transport activity and anchoring in the membrane. Transport kinetic analyses for the substrates sulfobromophthalein and taurocholate showed a loss of function for the L361>A mutant, whereas the transport activity was maintained by the L361>R mutant, indicating that the positive charge at position 361 is important for transport activity of OATP1B3. Comparative modeling with OATP1A2 and OATP2B1 revealed that the pore size around this lysine residue is larger in OATP1A2 and smaller in OATP2B1 compared with OATP1B3, which could be related to the respective substrate spectra. Cell surface expression of L399>A and L399>R was decreased to 16 and 72% compared with wild-type OATP1B3 (p < 0.001), respectively, indicating that the positive charge of lysine at position 399 is necessary for an unimpaired cell surface expression. Furthermore, we provide a summary of amino acids, which influence the transport activity of OATP1B3.     

Protein kinase C regulates the internalization and function of the human organic anion transporting polypeptide 1A2

BACKGROUND AND PURPOSE

The human organic anion transporting polypeptide 1A2 (OATP1A2) is expressed in cells from several regions of the human body, including the kidney, cholangiocytes and the blood-brain barrier, and mediates the cellular flux of various anionic substances, including drugs in clinical use. Several related mammalian transporters have been shown to be subject to post-translational regulation, including kinase-induced internalization. In the present study the role of protein kinase C (PKC) in the regulation of OATP1A2 was investigated in an in vitro cell model.

EXPERIMENTAL APPROACH

COS-7 cells in which OATP1A2 was overexpressed were treated with the PKC-specific activator (phorbol 12-myristate 13-acetate; PMA) and the PKC-specific inhibitor (Go6976). The impact of these treatments on the function and regulation of OATP1A2 was determined.

KEY RESULTS

PKC activation decreased the transport function of OATP1A2 in a time- and concentration-dependent manner. PMA (0.1 µM) decreased the V_max of oestrone-3-sulphate uptake and decreased the cell surface expression of OATP1A2 immunoreactive protein; these effects of PMA were prevented by the PKC specific inhibitor Go6976. In further studies, PMA treatment accelerated the internalization of OATP1A2 but did not affect its recycling. The disruption of clathrine-dependent endocytosis attenuated both the constitutive and PKC-modulated internalization of OATP1A2. In contrast, blocking the caveolin-dependent pathway was without effect.

CONCLUSIONS AND IMPLICATIONS

PKC regulates the transport function of OATP1A2 by modulating protein internalization; this effect of PKC is mediated in part by clathrine-dependent pathways.     

Cloning and functional characterization of the pig (Sus scrofa) organic anion transporting polypeptide 1a2

Abstract

1. Organic anion transporting polypeptides (OATPs) are a family of transporter proteins that have been extensively recognized as key determinants of absorption, distribution, metabolism and excretion of various drugs. Human OATP1A2 has been demonstrated to transport wide spectrum of endogenous and exogenous compounds. Study on OATP1A2 orthologues of other species, however, is still limited.

2. Here, we described the cloning and functional characterization of a member of the OATP/Oatp family member obtained from pig (Sus scrofa) liver. Sequence analysis suggested that it has a high homology with human OATP1A2 and bovine Oatp1a2. Prototypic substrates estrone-3-sulfate (E-3-S) and taurocholic acid were transported by the protein. The transport of these two substrates is pH-dependent, with lower pH showing higher uptake function. Kinetic study showed the transport of these two substrates have a K_m of 42.5?±?12.1 and 33.1?±?8.7?µM, respectively. Pig Slco1a2 has the highest expression level in the liver, and to a less extend in the brain and small intestine.

3. In conclusion, an OATP member was cloned from pig liver. Sequence analysis and phylogenic study revealed it as an orthologue of human OATP1A2. Its kinetic characteristic for prototypic substrates and organ distribution are similar with that of OATP1A2.     

The role of organic anion transporting polypeptides in drug absorption,distribution, excretion and drug-drug interactions

Introduction: The in vivo fate and effectiveness of a drug depends highly on its absorption, distribution, metabolism, excretion and toxicity (ADME-Tox). Organic anion transporting polypeptides (OATPs) are membrane proteins involved in the cellular uptake of various organic compounds, including clinically used drugs. Since OATPs are significant players in drug absorption and distribution, modulation of OATP function via pharmacotherapy with OATP substrates/inhibitors, or modulation of their expression, affects drug pharmacokinetics. Given their cancer-specific expression, OATPs may also be considered anticancer drug targets.

Areas covered: We describe the human OATP family, discussing clinically relevant consequences of altered OATP function. We offer a critical analysis of published data on the role of OATPs in ADME and in drug–drug interactions, especially focusing on OATP1A2, 1B1, 1B3 and 2B1.

Expert opinion: Four members of the OATP family, 1A2, 1B1, 1B3 and 2B1, have been characterized in detail. As biochemical and pharmacological knowledge on the other OATPs is lacking, it seems timely to direct research efforts towards developing the experimental framework needed to investigate the transport mechanism and substrate specificity of the poorly described OATPs. In addition, elucidating the role of OATPs in tumor development and therapy response are critical avenues for further research.