Potential role of organic anion transporting polypeptide 1B1 (OATP1B1) in the selective hepatic uptake of hematoporphyrin monomethyl ether isomers

Aim:

Hematoporphyrin monomethyl ether (HMME), which consists of equal amounts of isomers HMME-1 and HMME-2, is a novel porphyrin-related drug for photodynamic therapy. This study was aimed to investigate the uptake transporter-mediated selective uptake of HMME into the liver and to identify the major uptake transporter isoforms involved.

Methods:

Adult SD rats were intravenously injected with a single dose of HMME (5 mg/kg) with or without rifampicin (an inhibitor of organic anion transporting polypeptides OATP1B1 and OATP1B3, 25 mg/kg). Blood samples were collected, and HMME concentrations were measured using LC-MS/MS. Rat hepatocytes, human hepatocytes and HEK293 cells expressing OATP1B1, OATP1B3, or OATP2B1 were used to investigate the uptake of HMME or individual isomers in vitro.

Results:

Co-administration of rifampicin significantly increased the exposure of HMME isomers, and decreased the AUC ratio of HMME-1 to HMME-2 from 1.98 to 1.56. The uptake of HMME-2 into human hepatocytes and the HEK293 cells expressing OATP1B1 or OATP2B1 in vitro was 2–7 times greater than that of HMME-1, whereas OATP1B3 mediated a higher HMME-1 uptake. OATP1B1 exhibited a higher affinity for HMME-2 than for HMME-1 (the Km values were 0.63 and 5.61 μmol/L, respectively), which were similar to those in human hepatocytes. By using telmisartan (a non-specific OATP inhibitor) and rifampicin, OATP2B1 was demonstrated to account for <20% of hepatic HMME uptake.

Conclusion:

OATP1B1 is the major transporter involved in the rapid hepatic uptake of HMME, and the greater uptake of HMME-2 by OATP1B1 may lead to a lower exposure of HMME-2 than HMME-1 in humans.     

有机阴离子转运肽在药物转运中的作用

溶质运载蛋白家族(solute carrier family,SLC)和ATP结合盒转运蛋白家族(ATP binding cas-sette family,ABC)在药物吸收、消除和组织分布中起重要作用。本综述将对有机阴离子转运肽(or-ganic anion transporting polypeptide,OATP)的最新命名、分类、组织分布、功能及在药物转运中的作用加以介绍。     

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.     

Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family

1.?The organic anion transporting polypeptides (humans OATP; other species Oatp) belong to the SLCO gene superfamily of transporters and are twelve transmembrane domain glycoproteins expressed in various epithelial cells. Some OATPs/Oatps are expressed in a single organ, while others are expressed ubiquitously.

2.?The functionally characterized members mediate sodium-independent transport of a variety of structurally independent, mainly amphipathic organic compounds, including bile salts, hormones and their conjugates, toxins, and various drugs.

3.?This review summarizes the general features and the substrates of the eleven human OATPs. Furthermore, it reviews what is known about the mechanism of their multispecificity, their predicted structure, their role in drug–food interactions, and their role in cancer.

4.?Finally, some open questions are raised that need to be addressed to advance OATP research in the near future.     

有机阴离子转运多肽1B3的研究进展

有机阴离子转运多肽1B3(organic anion transporting polypeptide 1B3,OATP1B3)属于溶质转运体(solute carrier,SLC)超家族,主要负责将内、外源物质转运至肝细胞代谢。OATP1B3是肝脏特异性转运体,通常局限性地分布于肝细胞窦状隙侧肝细胞膜上,近期研究发现在前列腺癌、结肠癌、肺癌等肿瘤组织和细胞中也存在着高表达。溶质转运体1B3(SLCO1B3)具有明显的基因多态性,334T>G和699G>A单体型可明显影响OATP1B3的转运活性,从而介导药物-药物相互作用的发生,导致临床用药的个体差异。此外,OATP1B3可通过作用于孕烷X受体(pregnane X receptor,PXR)和组成性雄甾烷受体(constitutive androstane receptor,CAR)等核受体配体的转运,影响体内PXR和CAR的转录活性,从而调控药物代谢酶如细胞色素P450 3A4(CYP3A4)的表达。本文将对OATP1B3近年来的研究进展进行综述。     

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.     

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.     

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.     

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]     

Contribution of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to hepatic uptake of nateglinide, and the prediction of drug-drug interactions via these transporters

Objectives We have investigated the contributions of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to the hepatic uptake of nateglinide, and the possibility of drug–drug interactions via these transporters. Methods Uptake studies using transporter‐expressing HEK293 cells and cryopreserved human hepatocytes were performed to examine the contributions of each transporter. Inhibition studies using cryopreserved human hepatocytes were performed to examine the possibility of drug–drug interactions. Key findings The rate of saturable hepatic uptake of nateglinide using human hepatocytes was 47.6%. A certain increase in uptake was observed in the examination using transporter‐expressing HEK293 cells, indicating contributions of OATP1B1 and OATP1B3 to hepatic nateglinide uptake. The 50% inhibitory concentration (IC50) values of nateglinide using cryopreserved human hepatocytes for uptake of estrone 3‐sulfate (substrate of OATP1B1), and cholecystokinin octapeptide (substrate of OATP1B3) were 168 and 17.4 µmol/l, respectively. Moreover, ciclosporin inhibited saturable hepatic uptake of nateglinide with an IC50 value of 6.05 µmol/l. The calculated 1 + I_in,max,u/IC50 values for inhibition of OATP1B1 and OATP1B3 by nateglinide, and the inhibition of saturable uptake of nateglinide by ciclosporin, were all close to 1, indicating a low clinical risk of drug–drug interaction with nateglinide taken up via OATP1B1 and OATP1B3. Conclusions OATP1B1 and OATP1B3 may have contributed to the hepatic uptake of nateglinide, but the possibility of drug–drug interactions appeared to be low.     

6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响

目的 研究6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响。方法 体外培养稳定高表达OATP1B1和OATP1B1基因多态性A388G、T521C的人胚肾细胞（HEK293）株,高表达空白载体（Mock）的HEK293细胞为空白对照,实时荧光定量PCR（qRT-PCR）法检测各转运体细胞中mRNA表达;放射性标记化合物³Hestrone sulfate作为转运底物、利福平作为阳性抑制剂验证各高表达细胞的转运活性;测定30 μmol·L^-1的达比加群、辛伐他汀、替格瑞洛、卡培他滨、多西他赛、依那普利对各细胞³H-estrone sulfate摄入活性的抑制作用,并依据抑制试验结果,进一步测定辛伐他汀、替格瑞洛、多西他赛对转运体细胞的半数抑制浓度（IC₅₀）。结果 HEK293细胞内导入的各种转运体基因都呈现良好的复制表达;OATP1B1、OATP1B1/A388G、OATP1B1/T521C对底物³H-estrone sulfate（5 μmol·L^-1）的转运活性分别为Mock细胞的39、49和48倍,30 μmol·L^-1利福平添加后,可将细胞的转运活性抑制到50%以下;辛伐他汀、替格瑞洛、多西他赛对OATP1B1的抑制作用较强,30 μmol·L^-1给药的转运活性分别为对照组的（40.09±1.95）%、（33.82±0.61）%、（45.08±0.22）%;辛伐他汀对OATP1B1、OATP1B1/A388G、OATP1B1/T521C的IC₅₀分别为14.2、＞100、＞100 μmol·L^-1,替格瑞洛的IC₅₀分别为19.1、68.4、＞100 μmol·L^-1,多西他赛的IC₅₀分别为17.6、22.9、19.3 μmol·L^-1。结论 OATP1B1基因多态性在一定程度上改变了抑制剂对转运体活性的影响程度。     

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.     

Increased plasma concentrations of an antidyslipidemic drug pemafibrate co-administered with rifampicin or cyclosporine A in cynomolgus monkeys genotyped for the organic anion transporting polypeptide 1B1

In vitro permeability and in vivo pharmacokinetics of pemafibrate were investigated in human intestinal and animal models untreated or pretreated with cyclosporine A or rifampicin to evaluate any drug interactions. Ratios of basal to apical apparent permeability (P_app) over apical to basal P_app in the presence of pH gradients decreased from 0.37 to 0.080 on rifampicin co-incubation, suggesting active transport of pemafibrate from basal to apical sides in intestinal models. Plasma concentrations of intravenously administered pemafibrate were enhanced moderately in control mice but only marginally in humanized-liver mice by oral pretreatment with rifampicin [an organic anion transporting polypeptide (OATP) 1B1 inhibitor] 1 h before the administration of pemafibrate. In three cynomolgus monkeys genotyped as wild-type OATP1B1 (2 homozygous and 1 heterozygous), oral dosing of cyclosporine A 4 h or rifampicin 1 h before pemafibrate administration significantly increased the areas under the plasma concentration–time curves (AUC) of intravenously administered pemafibrate by 4.9- and 7.4-fold, respectively. Plasma AUC values of three pemafibrate metabolites in cynomolgus monkeys were also increased by cyclosporine A or rifampicin. These results suggested that pemafibrate was actively uptaken in livers and rapidly cleared from plasma in cynomolgus monkeys; this rapid clearance was suppressible by OATP1B1 inhibitors.     

Frequencies of single nucleotide polymorphisms and haplotypes of organic anion transporting polypeptide 1B1 SLCO1B1 gene in a Finnish population

Objective Organic anion transporting polypeptide 1B1 (OATP1B1) is a drug uptake transporter located at the sinusoidal membrane of hepatocytes. Our aim was to establish high-throughput genotyping assays for the major known single nucleotide polymorphisms (SNP) in the SLCO1B1 gene encoding OATP1B1 and to investigate the frequencies of SNPs and haplotypes of SLCO1B1 in a large Caucasian population.Methods Distribution of SLCO1B1 alleles was determined in 468 healthy Finnish Caucasian subjects at 11 variant sites spanning the entire gene by using allelic discrimination with TaqMan 5′ nuclease assays.Results The allelic frequencies of SLCO1B1 SNPs were 9.7% for g.−11187G>A, 0.4% for g.−11110T>G, 8.0% for g.−10499A>C, 46.2% for c.388A>G, 13.1% for c.411G>A, 13.1% for c.463C>A, 20.2% for c.521T>C, 53.2% for c.571T>C, 46.4% for c.597C>T, 4.0% for c.1929A>C and 48.8% for c.*439T>G. Altogether, 26 haplotypes were statistically inferred. The most common haplotype, with a frequency of 35.6%, contained variant allele C at position c.571, but had the reference nucleotide at all other positions investigated. The functionally significant c.521T>C SNP existed in four major haplotypes, which could be differentiated by the g.−11187G>A, g.−10499A>C and c.388A>G SNPs. The frequencies of these haplotypes were 7.9% for g.−11187G/g.−10499C/c.388G/c.521C (*16), 6.9% for AAGC (*17), 2.7% for GAAC (*5) and 2.4% for GAGC (*15).Conclusion Sequence variations of SLCO1B1 occur at high frequencies in the Caucasian population. Further studies are needed to characterise the effects of SLCO1B1 haplotypes, especially *16, *17, *15 and *5, on drug pharmacokinetics and response, and to determine the frequencies of SLCO1B1 SNPs and haplotypes in different populations.     

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.     

Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3

Background and purpose:

Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin.

Experimental approach:

Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated.

Key results:

Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V_max normalized to cell surface protein expression for Lys41>Ala (wild type: 190 ± 8, Lys41>Ala:16 ± 4 pmol (mg protein)⁻¹ min⁻¹, P < 0.001), whereas V_max of Lys41>Arg and Arg580>Lys (103 ± 8 and 123 ± 14 pmol (mg protein)⁻¹ min⁻¹, P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala.

Conclusions and implications:

We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.     

Effect of inhalation exposure to toluene on the activity of organic anion transporting polypeptide (Oatp) using pravastatin as a probe drug in rats

1.?Toluene, used as a pure substance or in solvent mixtures, is the cause of occupational exposures of large numbers of workers in the world. The organic anion transporting polypeptides (OATP: human; Oatp: rodents) are drug carriers which have been frequently associated to drug–drug interactions. The objective of this study was to evaluate the influence of inhalation exposure to toluene in Oatp in vivo activity using pravastatin as a probe drug in rats.

2.?Male Wistar rats ((n?=?6 per sampling time) were exposed to 85 mg/m³ toluene by inhalation or air in a nose only exposure system for 6 h/d, 5 d/week during 4 weeks, in order to simulate the occupational exposure to toluene at level slightly above the occupational exposure limit proposed by the American Conference of Governmental Industrial Hygienists (ACGIH). After 4 weeks of exposure, animals received a single dose of 20 mg/kg pravastatin orally.

3.?Areas under concentration × time curves extrapolated to infinite (AUC^0–∞) were calculated by Gauss Laguerre quadrature. Non-exposed animals showed AUC^0–∞ of 726.0 (261.8) ng h/mL for pravastatin and rats exposed to toluene 85 mg/m3 showed AUC^0–∞ of 681.8 (80.1)?ng?h/mL [data presented as mean (standard error of the mean)]. No significant difference was observed in pravastatin kinetic disposition between groups in terms of 95% confidence interval for the difference between means.

4.?Toluene exposure by inhalation did not change the in vivo activity of Oatp evaluated by pravastatin kinetic disposition in rats.     

Interaction characteristics of flavonoids with human organic anion transporter 1 (hOAT1) and 3 (hOAT3)

The present study aimed to investigate the interaction characteristics of flavonoids with human organic anion transporter 1 (hOAT1) and 3 (hOAT3). Five flavonoids (morin, silybin, naringin, naringenin and quercetin) were selected and their interaction characteristics with hOAT1 and hOAT3 were examined in MDCK cells overexpressing hOAT1 or hOAT3. Among tested flavonoids, morin and silybin exhibited significant inhibition effects on the cellular uptake of [3H]-para-aminohippuric acid ([3H]-PAH) in MDCK-hOAT1 cells with Ki of 0.46 microM and 24 microM, respectively, while all the tested flavonoids appeared to be less interactive with hOAT3 compared to hOAT1. A kinetic study suggested that morin and silybin inhibited hOAT1-mediated cellular uptake of [3H]-PAH in a competitive manner. Furthermore, morin and silybin were translocated by hOAT1 across the cellular membrane. In conclusion, the present study identified some of flavonoids as a new class of hOAT1 inhibitors, suggesting a potential for flavonoid-drug interactions via the modulation of hOAT1 activity.     

Recent advances in preclinical in vitro approaches towards quantitative prediction of hepatic clearance and drug-drug interactions involving organic anion transporting polypeptide (OATP) 1B transporters

Hepatic uptake mediated by organic anion transporting polypeptide (OATP) 1B1 and 1B3 can serve as a major elimination pathway for various anionic drugs and as a site of drug-drug interactions (DDIs). This article provides an overview of the in vitro approaches used to predict human hepatic clearance (CL_h) and the risk of DDIs involving OATP1Bs. On the basis of the so-called extended clearance concept, in vitro–in vivo extrapolation methods using human hepatocytes as in vitro systems have been used to predict the CL_h involving OATP1B-mediated hepatic uptake. CL_h can be quantitatively predicted using human donor lots possessing adequate OATP1B activities. The contribution of OATP1Bs to hepatic uptake can be estimated by the relative activity factor, the relative expression factor, or selective inhibitor approaches, which offer generally consistent outcomes. In OATP1B1 inhibition assays, substantial substrate dependency was observed. The time-dependent inhibition of OATP1B1 was also noted and may be a mechanism underlying the in vitro–in vivo differences in the inhibition constant of cyclosporine A. Although it is still challenging to quantitatively predict CL_h and DDIs involving OATP1Bs from only preclinical data, understanding the utility and limitation of the current in vitro methods will pave the way for better prediction.     

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.