Hepatic Uptake Transporter OATP1B1 Research Articles

Evaluation of the selectivity of several OATP1B biomarkers using relative activity factor method.

In recent years, some endogenous substrates of organic anion transporting polypeptide 1B (OATP1B) have been identified and characterized as potential biomarkers to assess OATP1B-mediated clinical drug-drug interactions (DDIs). However, quantitative determination of their selectivity to OATP1B are still limited. In this study, we developed a relative activity factor (RAF) method to determine the relative contribution of hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1, and sodium-taurocholate co-transporting polypeptide (NTCP) on hepatic uptake of several OATP1B biomarkers, including coproporphyrins I (CPI), CPIII, sulfate conjugates of bile acids: glycochenodeoxycholic acid sulfate (GCDCA-S), glycodeoxycholic acid sulfate (GDCA-S), and taurochenodeoxycholic acid sulfate (TCDCA-S). RAF values for OATP1B1, OATP1B3, OATP2B1, and NTCP were determined in cryopreserved human hepatocytes and transporter transfected cells using pitavastatin, cholecystokinin, resveratrol-3-O-β-D-glucuronide, and taurocholic acid (TCA) as reference compounds, respectively. OATP1B1-specific pitavastatin uptake in hepatocytes was measured in the absence and presence of 1 µM estropipate, while NTCP-specific TCA uptake was measured in the presence of 10 µM rifampin. Our studies suggested that CPI was a more selective biomarker for OATP1B1 than CPIII, while GCDCA-S and TCDCA-S were more selective to OATP1B3. OATP1B1 and OATP1B3 equally contributed to hepatic uptake of GDCA-S. The mechanistic static model, incorporating the fraction transported (ft) of CPI/III estimated by RAF and in vivo elimination data, predicted several perpetrator interactions with CPI/III. Overall, RAF method combined with pharmacogenomic and DDI studies is a useful tool to determine the selectivity of transporter biomarkers and facilitate the selection of appropriate biomarkers for DDI evaluation. Significance Statement We developed a new RAF method to quantitatively determine the contribution of hepatic uptake transporters OATP1B1, OATP1B3, OATP2B1, and NTCP on several OATP1B biomarkers (CPI, CPIII, GCDCA-S, GDCA-S, and TCDCA-S) and evaluated their predictivity on perpetrator-biomarker interactions. Our studies suggest that RAF method is a useful tool to determine the selectivity of transporter biomarkers. This method combined with pharmacogenomic and DDI studies will facilitate mechanistic interpretation and modeling of biomarker data and the selection of appropriate biomarkers for DDI evaluation.

Neither Gastric Bypass Surgery Nor Diet-Induced Weight-Loss Affect OATP1B1 Activity as Measured by Rosuvastatin Oral Clearance.

Rosuvastatin pharmacokinetics is mainly dependent on the activity of hepatic uptake transporter OATP1B1. In this study, we aimed to investigate and disentangle the effect of Roux-en-Y gastric bypass (RYGB) and weight loss on oral clearance (CL/F) of rosuvastatin as a measure of OATP1B1-activity. Patients with severe obesity preparing for RYGB (n = 40) or diet-induced weight loss (n = 40) were included and followed for 2 years, with four 24-hour pharmacokinetic investigations. Both groups underwent a 3-week low-energy diet (LED; < 1200 kcal/day), followed by RYGB or a 6-week very-low-energy diet (VLED; < 800 kcal/day). A total of 80 patients were included in the RYGB group (40 patients) and diet-group (40 patients). The weight loss was similar between the groups following LED and RYGB. The LED induced a similar (mean [95% CI]) decrease in CL/F in both intervention groups (RYGB: 16% [0, 31], diet: 23% [8, 38]), but neither induced VLED resulted in any further changes in CL/F. At Year 2, CL/F had increased by 21% from baseline in the RYGB group, while it was unaltered in the diet group. Patients expressing the reduced function SLCO1B1 variants (c.521TC/CC) showed similar changes in CL/F over time compared with patients expressing the wild-type variant. Neither body weight, weight loss nor RYGB per se seem to affect OATP1B1 activity to a clinically relevant degree. Overall, the observed changes in rosuvastatin pharmacokinetics were minor, and unlikely to be of clinical relevance.

A Metabolomics Approach for Predicting OATP1B-Type Transporter-Mediated Drug-Drug Interaction Liabilities.

In recent years, various endogenous compounds have been proposed as putative biomarkers for the hepatic uptake transporters OATP1B1 and OATP1B3 that have the potential to predict transporter-mediated drug–drug interactions (DDIs). However, these compounds have often been identified from top–down strategies and have not been fully utilized as a substitute for traditional DDI studies. In an attempt to eliminate observer bias in biomarker selection, we applied a bottom–up, untargeted metabolomics screening approach in mice and found that plasma levels of the conjugated bile acid chenodeoxycholate-24-glucuronide (CDCA-24G) are particularly sensitive to deletion of the orthologous murine transporter Oatp1b2 (31-fold increase vs. wild type) or the entire Oatp1a/1b(−/−)cluster (83-fold increased), whereas the humanized transgenic overexpression of hepatic OATP1B1 or OATP1B3 resulted in the partial restoration of transport function. Validation studies with the OATP1B1/OATP1B3 inhibitors rifampin and paclitaxel in vitro as well as in mice and human subjects confirmed that CDCA-24G is a sensitive and rapid response biomarker to dose-dependent transporter inhibition. Collectively, our study confirmed the ability of CDCA-24G to serve as a sensitive and selective endogenous biomarker of OATP1B-type transport function and suggests a template for the future development of biomarkers for other clinically important xenobiotic transporters.

Determination of the endogenous OATP1B biomarkers glycochenodeoxycholate-3-sulfate and chenodeoxycholate-24-glucuronide in human and mouse plasma by a validated UHPLC-MS/MS method

Glycochenodeoxycholate-3-sulfate (GCDCA-S) and chenodeoxycholate-24-glucuronide (CDCA-24G) are bile acid metabolites that potentially serve as endogenous biomarkers for drug-drug interactions mediated by the hepatic uptake transporters OATP1B1 and OATP1B3. We developed and validated a novel UHPLC-MS/MS method for the quantitative determination of GCDCA-S and CDCA-24G in mouse and human plasma with a lower limit of quantitation of 0.5 ng/mL. Chromatographic separation was achieved on an Accucore aQ column (50 mm × 2.1 mm, dp = 2.6 μm) maintained at 20 °C and a gradient mobile phase comprising 2 mM ammonium acetate in water and methanol. The extraction recoveries of GCDCA-S and CDCA-24G were >80 %, and linear (r2 > 0.99) calibration curves ranged 0.5–100 ng/mL (CDCA-24G and GCDCA-S in mouse plasma) or 0.5–1000 ng/mL (GCDCA-S in mouse plasma). Values for precision (CV < 11.6 %) and accuracy bias (10.9 %) of analyte-spiked quality control samples verified that water was an acceptable matrix to prepare calibrators. This method was successfully applied to establish baseline activity of OATP1B1/OATP1B3 in humans and mice and establish the in vivo effects of OATP1B1/OATP1B3 inhibitors rifampin and micafungin.

Minimal contribution of the hepatic uptake transporter OATP1B1 to the inter-individual variability in SN-38 pharmacokinetics in cancer patients without severe renal failure.

SN-38, a pharmacologically active metabolite of irinotecan, is taken up into hepatocytes by organic anion transporting polypeptide (OATP) 1B1. The effects of functional OATP1B1 521T>C on the pharmacokinetics of SN-38 remain controversial. Here, we prospectively examined the effects of OATP1B1 function on the area under the plasma total or unbound concentration-time curve (tAUC or uAUC) of SN-38 by assessing OATP1B1 521T>C and the plasma levels of endogenous OATP1B1 substrates, coproporphyrin (CP)-I and III, in cancer patients treated with irinotecan. We enrolled cancer patients who were treated with an irinotecan-containing regimen and did not have severe renal failure. The total and unbound concentrations of SN-38 in the plasma were measured by high-performance liquid chromatography. AUC values were calculated and normalized to the actual irinotecan dose (AUC/dose). The OATP1B1 521T>C was analyzed by direct sequencing. Concentrations of the endogenous substrates in plasma before irinotecan treatment (baseline) were determined by liquid chromatography with tandem mass spectrometry. Twenty-two patients with a median estimated glomerular filtration rate of 74.8mL/min (range 32.6-99.6) were examined. Both tAUC/dose and uAUC/dose were associated with the grade of neutropenia; however, they were not associated with OATP1B1 521T>C or baseline CP-I and III levels. It is worth noting that these baseline concentrations were significantly higher in patients with OATP1B1 521C, supporting functional changes in OATP1B1. The contribution of OATP1B1 activity to inter-patient variability in the systemic exposure to SN-38 is likely minimal in patients without severe renal failure.

Interaction of the FLT3 Inhibitor Gilteritinib with Xenobiotic Uptake Transporters

Objective: Gilteritinib is a kinase inhibitor approved for relapsed/refractory FLT3-mutated acute myeloid leukemia. The pharmacokinetic properties of gilteritinib are characterized by extensive differences in exposure between patients receiving the same dose, and this high degree of variability may have important toxicological ramification. Gilteritinib is believed to be primarily eliminated via CYP3A4-mediated metabolism but mechanisms of hepatocellular uptake and its interaction with xenobiotic transporters remain poorly understood. Here, we re-examined the contribution of CYP3A-mediated elimination to gilteritinib exposure and its transporter-mediated drug-drug interaction potential. Methods: Gilteritinib transport was studied in vitro using HEK293 cells stably transfected with human OATP1B1, its murine orthologue OATP1B2, OCT1, OCT2, or MATE1. Uptake studies were performed to evaluate the effect of gilteritinib on uptake of the radiolabeled substrates estradiol-17β-d-glucuronide, tetraethylammonium, and metformin, followed by liquid scintillation counting. Pharmacokinetic studies were performed with a single oral dose of gilteritinib (10 mg/kg; n=5 per group) in adult wild-type mice and sex- and age-matched mice that are CYP3A-deficient or OATP1B2-deficient, via repeat blood sampling (6 samples over 8 hours). Concentrations of gilteritinib were determined using a validated LC-MS/MS method, and pharmacokinetic parameters were calculated by non-compartmental analysis using Phoenix WinNonlin v8.0. Results: Compared to wild-type mice, the systemic exposure to gilteritinib was not significantly altered in CYP3A-deficient mice (1423 ± 142 vs 1468 ± 147 ng.h/mL), suggesting that the clearance of gilteritinib in mice is largely independent of CYP3A function. In vitro studies demonstrated that gilteritinib is an inhibitor of the hepatic uptake transporter OATP1B1 (IC50, 2.23 µM), suggesting that gilteritinib elimination may be dependent on OATP1B-genotype status. This hypothesis was verified by the demonstration that the peak concentration and area under the curve were increased by 2.5- and 2.2-fold, respectively, in OATP1B2-deficient mice compared with wild-type mice. In addition, we found that gilteritinib is a potent inhibitor of the hepatic transporter OCT1 (IC50, 300 nM) as well as OCT2 (IC50, 150 nM) and MATE1 (IC50, 5 nM), transporters involved in the renal tubular excretion of cationic drugs. Conclusions: This study suggests that CYP3A-deficiency is not associated with a profound phenotypic change in the pharmacokinetic handling of gilteritinib in mice. This unexpected finding suggests that the contribution of CYP3A-mediated metabolism to previously reported drug-drug interactions of gilteritinib with CYP3A4-inhibitory azole antifungals, such as itraconazole, is low and may instead be mechanistically linked with OATP1B-mediated transport modulation. This possibility is currently under further investigation in a transgenic mouse model with hepatic expression of human OATP1B1.

Role of OATP1B1 and OATP1B3 in Drug-Drug Interactions Mediated by Tyrosine Kinase Inhibitors.

Failure to recognize important features of a drug’s pharmacokinetic characteristics is a key cause of inappropriate dose and schedule selection, and can lead to reduced efficacy and increased rate of adverse drug reactions requiring medical intervention. As oral chemotherapeutic agents, tyrosine kinase inhibitors (TKIs) are particularly prone to cause drug-drug interactions as many drugs in this class are known or suspected to potently inhibit the hepatic uptake transporters OATP1B1 and OATP1B3. In this article, we provide a comprehensive overview of the published literature and publicly-available regulatory documents in this rapidly emerging field. Our findings indicate that, while many TKIs can potentially inhibit the function of OATP1B1 and/or OATP1B3 and cause clinically-relevant drug-drug interactions, there are many inconsistencies between regulatory documents and the published literature. Potential explanations for these discrepant observations are provided in order to assist prescribing clinicians in designing safe and effective polypharmacy regimens, and to provide researchers with insights into refining experimental strategies to further predict and define the translational significance of TKI-mediated drug-drug interactions.

Real-life management of drug-drug interactions between antiretrovirals and statins.

PIs cause drug-drug interactions (DDIs) with most statins due to inhibition of drug-metabolizing enzymes and/or the hepatic uptake transporter OATP1B1, which may alter the pharmacodynamic (PD) effect of statins. To assess the management of DDIs between antiretrovirals (ARVs) and statins in people living with HIV (PLWH) considering statin plasma concentrations, compliance with dosing recommendations and achievement of lipid targets. PLWH of the Swiss HIV Cohort Study were eligible if they received a statin concomitantly with ARVs. HDL, total cholesterol (TC) and statin plasma concentration were measured during follow-up visits. Individual non-HDL and TC target values were set using the Framingham score and the 2018 European AIDS Clinical Society recommendations. Data were analysed for rosuvastatin (n = 99), atorvastatin (n = 92), pravastatin (n = 46) and pitavastatin (n = 21). Rosuvastatin and atorvastatin underdosing frequently led to suboptimal PD response. Insufficient lipid control was observed with PIs despite high atorvastatin concentrations, likely explained by inhibition of OATP1B1 resulting in less statin uptake in the liver. Target lipid values were more often achieved with unboosted integrase inhibitors due to both their favourable DDI profiles and neutral effect on lipids. Insufficient lipid control was common with pravastatin and pitavastatin regardless of co-administered ARVs and despite using maximal recommended statin doses. The latter suggests lower efficacy compared with rosuvastatin or atorvastatin. Suboptimal management of DDIs with statin underdosing was observed in 29% of prescriptions. Integrase inhibitor-based regimens and/or treatment with rosuvastatin or atorvastatin should be favoured in patients with refractory dyslipidaemia.

Analysis of amino acid residues in the predicted transmembrane pore influencing transport kinetics of the hepatic drug transporter organic anion transporting polypeptide 1B1 (OATP1B1)

The hepatic uptake transporters OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) mediate the uptake of endogenous metabolites and drugs from blood into hepatocytes. Alterations of transport function are accompanied with variations in drug plasma concentrations and the risk of adverse drug effects. Thus, knowledge on amino acids determining substrate recognition or transport kinetics are important to predict alterations in transport kinetics. Therefore, we analyzed the charged amino acids His54 and Tyr169, both located at the extracellular entry of the predicted transmembrane pore of OATP1B1.Based on a computational analysis we established HEK293 cell lines overexpressing the mutant OATP1B1 proteins HEK-OATP1B1p.H54Q, -p.H54A, -p.Y169H and -p.Y169A and analyzed protein expression, localization and transport kinetics of the four OATP1B1 substrates bromosulfophthalein, estradiaol-17β-glucuronide, taurocholate and pravastatin.Consequences on transport were detected for all mutants and these were different for each amino acid exchange and for each substrate tested. For example, the exchange H54Q resulted in reduced transport for BSP (78% of wildtype OATP1B1 transport at 0.05μM, P<0.01) with reduced affinity to this substrate (Km value increases from 0.76μM to 8.04μM) but in stimulated E217βG transport (138% compared to wildtype transport at 10μM, P<0.001).Investigating amino acid exchanges located at the extracellular entry of the transport pore of the OATP1B1 protein we demonstrated that these residues are involved in modulating transport kinetics and this participation strongly depends on the substrate and not on the physicochemical character of the investigated amino acid.

The Nonmetabolized β-Blocker Nadolol Is a Substrate of OCT1, OCT2, MATE1, MATE2-K, and P-Glycoprotein, but Not of OATP1B1 and OATP1B3

Nadolol is a nonmetabolized β-adrenoceptor antagonist and is a substrate of OATP1A2, but not of OATP2B1. However, other drug transporters involved in translocation of nadolol have not been characterized in detail. We therefore investigated nadolol as a potential substrate of the hepatic uptake transporters OATP1B1, OATP1B3, and OCT1 and of the renal transporters OCT2, MATE1, and MATE2-K expressed in HEK cells. Moreover, the importance of P-glycoprotein (P-gp) for nadolol transport was studied using double transfected MDCK-OCT1-P-gp cells. Nadolol was not transported by OATP1B1 and OATP1B3. In contrast, a significantly higher nadolol accumulation (at 1 and 10 μM) was found in OCT1, OCT2, MATE1, and MATE2-K cells compared to control cells (P < 0.01). Km values for OCT2-, MATE1-, and MATE2-K-mediated nadolol uptake were 122, 531, and 372 μM, respectively. Cimetidine (100 μM, P < 0.01) and trimethoprim (100 μM, P < 0.001) significantly inhibited OCT1-, OCT2-, MATE1-, and MATE2-K-mediated nadolol transport. The P-gp inhibitor zosuquidar significantly reduced basal to apical nadolol transport in monolayers of MDCK-OCT1-P-gp cells. In summary, nadolol is a substrate of the cation transporters OCT1, OCT2, MATE1, MATE2-K, and of P-gp. These data will aid future in vivo studies on potential transporter-mediated drug-drug or drug-food interactions with involvement of nadolol.

Abstract 3696: Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations

Abstract Background: Several tyrosine kinase inhibitors (TKIs) evaluated in combination regimens with docetaxel, including axitinib, pazopanib, and sorafenib, can increase the systemic exposure to taxane in cancer patients by an unknown mechanism. We previously reported that differential expression of organic anion transporting polypeptides of the OATP1B-family in the human liver regulate the initial, rate-limiting step in the elimination of docetaxel, in advance of metabolism. In the current study, we tested the hypothesis that these interactions are mediated by the hepatic uptake transporter OATP1B1 using in vitro and in vivo models. Materials and Methods: The influence of 16 approved TKIs on transport was studied in vitro using HEK293 cells expressing OATP1B1 or its mouse equivalent OATP1B2, determined by assessing the effect of TKIs on the intracellular accumulation of estradiol-17β-D-glucuronide, or docetaxel. Contribution of this process to an interaction with docetaxel in vivo was determined by pharmacokinetic studies using mice that are knock-out for OATP1B2 or knock-in for OATP1B1. Results: Axitinib, nilotinib, pazopanib, and sorafenib were identified as potent inhibitors of OATP1B1 (&amp;gt;90% inhibition) when using estradiol-17β-D-glucuronide as a prototypical model substrate. As a representative of this class of TKIs, sorafenib was further evaluated and found to also potently inhibit OATP1B1-mediated transport of docetaxel with a half-inhibitory maximum concentration (IC50) of 6.96 nM, and almost completely inhibited the function of OATP1B1 and OATP1B2 at a concentration readily achievable in humans and mice. While OATP1B2-deficiency was associated with significantly increased exposure to docetaxel, as measured by peak plasma concentration (Cmax, P&amp;lt;0.05) and area under the curve (AUC, P&amp;lt;0.001), co-administration of a single or multiple oral dose of sorafenib did not result in statistically significantly altered exposure to docetaxel in either wildtype or OATP1B2 (-/-) mice. Conclusion: These findings support a direct contribution of OATP1B1 in previously recorded pharmacokinetic interactions between TKIs and docetaxel, which can be predicted from a simple in vitro experiment. Our failure to reproduce this interaction in mice suggests that caution is warranted when attempting to extrapolate in vivo findings to a clinical scenario. Citation Format: Shuiying Hu, Peter De Bruijn, Ron H.j. Mathijssen, Sharyn D. Baker, Alex Sparreboom. Inhibition of OATP1B1 by tyrosine kinase inhibitors: in vitro-in vivo correlations. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3696. doi:10.1158/1538-7445.AM2014-3696

Genetics is a major determinant of expression of the human hepatic uptake transporter OATP1B1, but not of OATP1B3 and OATP2B1

BackgroundOrganic anion transporting polypeptide (OATP) 1B1, OATP1B3, and OATP2B1 (encoded by SLCO1B1, SLCO1B3, SLCO2B1) mediate the hepatic uptake of endogenous compounds like bile acids and of drugs, for example, the lipid-lowering atorvastatin, thereby influencing hepatobiliary elimination. Here we systematically elucidated the contribution of SLCO variants on expression of the three hepatic OATPs under consideration of additional important covariates.MethodsExpression was quantified by RT-PCR and immunoblotting in 143 Caucasian liver samples. A total of 109 rare and common variants in the SLCO1B3-SLCO1B1 genomic region and the SLCO2B1 gene were genotyped by MALDI-TOF mass spectrometry and genome-wide SNP microarray technology. SLCO1B1 haplotypes affecting hepatic OATP1B1 expression were associated with pharmacokinetic data of the OATP1B1 substrate atorvastatin (n = 82).ResultsExpression of OATP1B1, OATP1B3, and OATP2B1 at the mRNA and protein levels showed marked interindividual variability. All three OATPs were expressed in a coordinated fashion. By a multivariate regression analysis adjusted for non-genetic and transcription covariates, increased OATP1B1 expression was associated with the coding SLCO1B1 variant c.388A > G (rs2306283) even after correction for multiple testing (P = 0.00034). This held true for haplotypes harboring c.388A > G but not the functional variant c.521T > C (rs4149056) associated with statin-related myopathy. c.388A > G also significantly affected atorvastatin pharmacokinetics. SLCO variants and non-genetic and regulatory covariates together accounted for 59% of variability of OATP1B1 expression.ConclusionsOur results show that expression of OATP1B1, but not of OATP1B3 and OATP2B1, is significantly affected by genetic variants. The SLCO1B1 variant c.388A > G is the major determinant with additional consequences on atorvastatin plasma levels.

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 (Ki 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.

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 [(3) H]UDCA and [(14) C]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 (1μM, 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.

A LC–MS/MS method to evaluate the hepatic uptake of the liver-specific magnetic resonance imaging contrast agent gadoxetate (Gd-EOB-DTPA) in vitro and in humans

Gadoxetate (Gd-EOB-DTPA, Primovist®) is a frequently used liver-specific magnetic resonance imaging (MRI) contrast agent which disposition is so far not fully understood in humans. Here, we describe the development and validation of a selective and sensitive quantification method to measure cellular in vitro concentrations as well as human serum concentrations of gadoxetate. The drug was measured after protein precipitation with acetonitrile and ethyl acetate-mediated sample concentration using amoxicillin as internal standard and liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) for detection. Hydrophilic interaction chromatography (HILIC) was performed by using the column Atlantis® HILIC Silica (2.1mm×100mm), a step-elution gradient with acetonitrile and ammonium acetate (5mM, pH 3.8) as mobile phases and a flow rate of 200μl/min. The MS/MS detection was done in the negative multiple reaction monitoring (MRM) mode by monitoring the m/z transitions 681.3/635.2 for gadotrexate and 363.8/222.7 for the internal standard. The method was validated between 5 and 4000ng/ml in serum and between 1.25 and 500ng/ml in cell lysates. The method was shown to possess sufficient specificity, accuracy, precision and stability without any matrix effects, thereby fulfilling current bioanalytical guidelines. The developed assay was successfully applied to quantify gadoxetate in cellular uptake studies in OATP1B1-transfected cell lines and to monitor serum concentrations-time profiles from a clinical pilot study performed in healthy volunteers carrying the wild-type or the functionally relevant variants T521C (*5) and A388G (*1b) of the hepatic uptake transporter OATP1B1.

SLCO1B1 genetic polymorphism influences mycophenolic acid tolerance in renal transplant recipients

This study aimed to determine the influence of gene candidates on mycophenolic acid (MPA) response during the first year of renal transplantation. A total of 218 renal transplant recipients who received MPA from the first day of transplantation at a fixed dose of 2 g/day were genotyped for ABCB1, ABCC2, UGT2B7, UGT1A9, SLCO1B1, SLCO1B3 and IMPDH1 polymorphisms. Clinical end points were MPA-related adverse drug reactions (ADRs) and acute rejection episodes during the first year post-transplantation. After correction for multiple statistical testing, SLCO1B1 (encoding the hepatic uptake transporter OATP1B1) was the only gene associated with MPA-related ADRs, showing a 75% risk reduction in favor of a protective effect of the SLCO1B1*5 allele (p = 0.002). In vitro experiments showed that MPA metabolites MPA-phenyl-glucuronide and MPA-acyl-glucuronide are substrates of OATP1B1. Their transport was decreased in the presence of the variant transporter (OATP1B1*5). These results suggest for the first time that carriers of the SLCO1B1*5 allele seem to be protected from MPA-related ADRs.

The metabolism and disposition of a potent inhibitor of hepatitis C virus NS3/4A protease

Compound A ((1aR,5S,8S,10R,22aR)-5-tert-butyl-N-{(1R,2S)-1-[(cyclopropylsulfonyl)carbamoyl]-2-ethenylcyclopropyl}-14-methoxy-3,6-dioxo-1,1a,3,4,5,6,9,10,18,19,20,21,22,22a-tetradecahydro-8H-7,10-methanocyclopropa[18,19][1,10,3,6]dioxadiazacyclononadecino[12,11-b]quinoline-8-carboxamide) is a prototype of a series of subnanomolar inhibitors of genotypes 1, 2, and 3 hepatitis C virus (HCV) NS3/4A proteases. HCV NS3/4A protease inhibitors have demonstrated high antiviral effects in patients with chronic HCV infection and are likely to form a key component of future HCV therapy.Compound A showed excellent liver exposure in rats, which is essential for compounds intended to treat HCV. The compound was mainly eliminated intact in bile and showed greater than dose proportional systemic exposure in rats. Compound A demonstrated time- and temperature-dependent uptake into rat and human hepatocytes and proved to be a substrate for rat hepatic uptake transporter Oatp1b2 and for human hepatic uptake transporters OATP1B1 and OATP1B3. The liver selectivity observed for this compound is likely to be due to transporter-mediated hepatic uptake together with moderate passive permeability.Metabolism was mainly CYP3A-mediated and generated a reactive epoxide on the vinylcyclopropyl sulfonamide moiety that could be quenched by glutathione. Similar metabolic profiles of Compound A were obtained in liver microsomes of rats and humans. The oral bioavailability at 5 mg/kg was low due to extensive hepatic first-pass effect but clearly the intestinal absorption was enough to deliver a high amount of the compound to the liver. The metabolism and disposition properties of Compound A are particularly attractive to support its evaluation as a drug candidate for the treatment of hepatitis C.

Cyclosporine A Increases Plasma Concentrations and Effects of Repaglinide

To the Editor: We read with interest the article by Türk et al. titled 'Repaglinide in the Management of New-Onset Diabetes Mellitus After Renal Transplantation' (1Türk T Pietruck F Dolff S et al.Repaglinide in the management of new-onset diabetes mellitus after renal transplantation..Am J Transplant. 2006; 6: 842-846Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). While we agree with the conclusions of this observational study that repaglinide can be effective in the treatment of new-onset diabetes mellitus (NODM) after renal transplantation, we are concerned about the risk of interaction between cyclosporine A and repaglinide. In their discussion, the authors suggest that relevant changes in repaglinide concentrations due to interactions with calcineurin inhibitors are unlikely to occur (1Türk T Pietruck F Dolff S et al.Repaglinide in the management of new-onset diabetes mellitus after renal transplantation..Am J Transplant. 2006; 6: 842-846Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). This suggestion is based on the dual metabolism of repaglinide by the cytochrome P450 (CYP) 2C8 and 3A4 isoenzymes, which are proposed to be able to compensate for each other if the other isoenzyme is inhibited (2Bidstrup TB Bjørnsdottir I Sidelmann UG Thomsen MS Hansen KT CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide..Br J Clin Pharmacol. 2003; 56: 305-314Crossref PubMed Scopus (173) Google Scholar). However, the fibric acid derivative gemfibrozil increases the area under the plasma concentration-time curve (AUC) of repaglinide (reflecting total exposure to repaglinide) almost 10-fold (3Niemi M Backman JT Neuvonen M Neuvonen PJ Effects of gemfibrozil,itraconazole, and their combination on the pharmacokinetics and pharmacodynamics of repaglinide: Potentially hazardous interaction between gemfibrozil and repaglinide..Diabetologia. 2003; 46: 347-351Crossref PubMed Scopus (241) Google Scholar), indicating that the dual metabolism of repaglinide does not eliminate the risk of interaction. Moreover, our recent results demonstrate that cyclosporine A markedly raises the plasma concentrations of repaglinide (4Kajosaari LI Niemi M Neuvonen M Laitila J Neuvonen PJ Backman JT Cyclosporine markedly raises the plasma concentrations of repaglinide..Clin Pharmacol Ther. 2005; 78: 388-399Crossref PubMed Scopus (177) Google Scholar). In the cross-over study in healthy adults, cyclosporine A (100 mg, ingested 13 h and 1 h before repaglinide) increased the peak plasma concentration of repaglinide 1.8-fold and its AUC 2.4-fold (4Kajosaari LI Niemi M Neuvonen M Laitila J Neuvonen PJ Backman JT Cyclosporine markedly raises the plasma concentrations of repaglinide..Clin Pharmacol Ther. 2005; 78: 388-399Crossref PubMed Scopus (177) Google Scholar). The extent of the interaction showed considerable interindividual variability (e.g. a 5.3-fold increase in repaglinide exposure was observed in one of the 12 subjects), which is, at least partially, due to genetic factors. Because the effects of cyclosporine A are dependent on its concentrations, the extent of this interaction can be greater in patients receiving continuous immunosuppression with cyclosporine A than in the study with a relatively small dose of cyclosporine A. A likely explanation for this interaction is the dual inhibitory effect of cyclosporine A on the CYP3A4-catalyzed biotransformation and active transporter-mediated uptake of repaglinide into hepatocytes. In addition to the well-known inhibitory effects of cyclosporine A on CYP3A4 and P-glycoprotein, cyclosporine A is a potent inhibitor of the hepatic uptake transporter OATP1B1 (previously known as LST-1, OATP2 or OATP-C) (5Shitara Y Itoh T Sato H Li AP Sugiyama Y Inhibition of transportermediated hepatic uptake as a mechanism for drug-drug interaction between cerivastatin and cyclosporin A..J Pharmacol Exp Ther. 2003; 304: 610-616Crossref PubMed Scopus (314) Google Scholar). Inhibition of this transporter partially explains the effects of cyclosporine A on the pharmacokinetics of other drugs, including repaglinide and several statins. Also gemfibrozil has a dual inhibitory effect, which explains its strong interaction with repaglinide: it inhibits both CYP2C8 and OATP1B1 (6Shitara Y Hirano M Sato H Sugiyama Y Gemfibrozil and its glucuronide inhibit the organic anion transporting polypeptide2 (OATP2/OATP1B1:SLC21A6)-mediated hepatic uptake and CYP2C8-mediated metabolism of cerivastatin: Analysis of the mechanism of the clinically relevant drug-drug interaction between cerivastatin and gemfibrozil..J Pharmacol Exp Ther. 2004; 311: 228-236Crossref PubMed Scopus (373) Google Scholar). The interaction between cyclosporine A and repaglinide is of clinical significance; it can increase the glucose-lowering efficacy of repaglinide, but it can also lead to an elevated risk of hypoglycemia. In the study of Türk et al., a successful blood glucose response to repaglinide was obtained in 86% of patients on cyclosporine A, while less than 60% of the other patients responded successfully to repaglinide (1Türk T Pietruck F Dolff S et al.Repaglinide in the management of new-onset diabetes mellitus after renal transplantation..Am J Transplant. 2006; 6: 842-846Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It is possible that the good response to repaglinide in cyclosporine A-treated patients was due to the pharmacokinetic interaction between these two agents. Moreover, Trk et al. report that three patients reported symptoms of a hypoglycemic episode during treatment with repaglinide (1Türk T Pietruck F Dolff S et al.Repaglinide in the management of new-onset diabetes mellitus after renal transplantation..Am J Transplant. 2006; 6: 842-846Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). It would be pertinent to know if these patients were also treated with cyclosporine A. To conclude, cyclosporine A can markedly increase the plasma concentrations of repaglinide. Their concomitant use may enhance the blood glucose-lowering effect of repaglinide, but this positive effect is associated with an increased risk of hypoglycemia. Therefore, care is warranted when using repaglinide in patients receiving cyclosporine A immunosuppression.

Fexofenadine pharmacokinetics are associated with a polymorphism of the SLCO1B1 gene (encoding OATP1B1)

The H1-receptor antagonist fexofenadine is a P-glycoprotein substrate [1]. However, the effects of genetic polymorphisms of the MDR1 (ABCB1) gene, which codes for P-glycoprotein on the disposition of fexofenadine are unclear [2, 3]. In addition, fexofenadine is a substrate of the intestinal uptake transporters organic anion transporting polypeptide 1A2 (OATP1A2, OATP-A) and 2B1 (OATP2B1, OATP-B) [1, 4]. Fruit juices such as grapefruit, orange and apple juice may decrease the oral bioavailability of fexofenadine by inhibiting the activity of OATP1A2 [5]. There seems to be no published data on whether fexofenadine is a substrate of the hepatic uptake transporter OATP1B1 (also known as OATP-C and OATP2), encoded by SLCO1B1. It has been recently demonstrated that certain single-nucleotide polymorphisms (SNP) of SLCO1B1 are associated with a decreased function of OATP1B1 in vitro. For example healthy carriers of the 521T > C allele [6] have an increased systemic exposure to pravastatin [7]. The aim of the present study was to characterize possible relationships between polymorphisms in the SLCO1B1 gene and the pharmacokinetics of fexofenadine in healthy Caucasian subjects. To this end, 20 Caucasian volunteers who had previously participated in a pharmacokinetic study with fexofenadine [3] were genotyped by TaqMan® 5′nuclease assays for the two SLCO1B1 SNPs that were significantly associated with increased pravastatin concentrations in our previous study [7], 521T > C (Val174Ala) and for the promoter SNP −11187G > A. Of the 20 subjects, two were homozygous and eight were heterozygous for the SLCO1B1521T > C SNP, the remaining 10 subjects having the reference genotype (521TT). Two subjects were heterozygous for the SLCO1B1−11187G > A polymorphism, and the remaining subjects had the reference (−11187GG) genotype. Both heterozygotes for the −11187G > A SNP were also heterozygous for the 521T > C SNP. In the two subjects with the 521CC (and −11187GG) genotype, the mean total AUC of fexofenadine was 76.0% higher (P C polymorphism had no significant influence on the Cmax or t1/2 of fexofenadine. There was a trend (P = 0.13) towards a gene-dose effect regarding the influence of the 521T > C SNP on the apparent nonrenal clearance of fexofenadine. Thus, it was lowest (389 ± 185 ml min−1) in subjects with the 521CC genotype, intermediate (775 ± 401 ml min−1) in those with the 521TC genotype and highest in those with the reference genotype (957 ± 325 ml min−1). Figure 1 Mean (± SEM) plasma concentrations of fexofenadine in 20 healthy subjects with different SLCO1B1 521T > C genotypes after a single 180 mg oral dose of fexofenadine HCl. SLCO1B1 521CC subjects (n = 2) (▴); SLCO1B1 521TC subjects ... A stepwise multiple regression analysis of data from subjects carrying the SLCO1B1521T > C and −11187G > A SNPs and the MDR13435C > T (exon 26), 2677G > T/A (exon 21), 1236C > T (exon 12) and 61 A > G (exon 2) SNPs indicated that only the SLCO1B1521T > C SNP was an independent predictor of the total AUC of fexofenadine (P T and 3435C > T were assigned into haplotype pairs according to Johne et al. [8] and data from subjects with these haplotypes were put into a stepwise multiple regression analysis with the SLCO1B1 SNP data. It was found that the MDR1 haplotypes had no independent effect on the AUC of fexofenadine (haplotype 2677T/3435C was present in one subject only). The results of this retrospective study demonstrated that the pharmacokinetics of fexofenadine were influenced by the SLCO1B1 genotype. Fexofenadine has been previously shown to be a substrate of OATP1A2 [1] and OATP2B1 [4]. Tamai et al. reported that OATP1A2 was not expressed in a single sample of human small intestine as analyzed by RT-PCR [9]. However, more recently, expression of OATP1A2 in human intestine was demonstrated [10], a finding supported indirectly by the work of Dresser et al. [5]. These investigators showed that certain fruit juices at 5% of normal strength markedly inhibited OATP1A2 mediated fexofenadine uptake in vitro and that the same juices considerably decreased the oral bioavailability of fexofenadine in vivo. A nonsynonymous SNP associated with a markedly impaired capacity for mediating the cellular uptake of fexofenadine was recently identified within the coding region of the SLCO1A2 gene (encoding for OATP1A2) [10]. Expression of OATP2B1 has been shown immunohistochemically at the apical membrane of human intestinal epithelial cells [11]. Known functionally significant SLCO2B1 SNPs are rare in Caucasians [7], and SLCO2B1 genotypes were not determined in the present study. OATP1B1 is exclusively expressed in the liver [12, 13]. That the SLCO1B1−11187G > A and 521T > C SNPs are associated with a higher pravastatin AUC [7] may be explained by decreased hepatic uptake of the drug, resulting in its slower elimination and lower systemic clearance. The pharmacokinetics of pravastatin and fexofenadine are similar in that these drugs do not undergo significant CYP-mediated biotransformation and are eliminated from the body unchanged mainly by biliary and urinary excretion [14, 15]. The influence of the SLCO1B1521T > C polymorphism on the AUC of fexofenadine suggests that the OATP1B1-mediated uptake of the drug into the liver is a prerequisite for its biliary elimination. This hypothesis is also supported by the trend towards a lower apparent nonrenal clearance of fexofenadine in subjects with the 521CC or 521TC genotype compared with those with the reference genotype. Studies on the effect of the 3435C > T SNP in the exon 26 of MDR1 on intestinal P-glycoprotein expression and on the disposition of P-glycoprotein substrates, including fexofenadine, have yielded conflicting results [16]. These might be partly explained by the influence of the SLCO1B1 genotype on fexofenadine disposition [2, 3]. In conclusion, we have demonstrated that the pharmacokinetics of fexofenadine are associated with a polymorphism of the SLCO1B1 gene (encoding OATP1B1). Further studies are required to characterize the interaction between fexofenadine and the hepatic uptake transporter OATP1B1.