Stefan Oswald

Hepatic Uptake of the Magnetic Resonance Imaging Contrast Agent Gd-EOB-DTPA: Role of Human Organic Anion Transporters

Contrast-enhancing magnetic resonance imaging with the liver-specific agent gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) has been shown to improve the detection rate of focal lesions. There is evidence from preclinical studies that multidrug organic anion transporters are involved in hepatic uptake of Gd-EOB-DTPA. Therefore, we evaluated affinity of the contrast agent to human organic anion-transporting polypeptides (OATP1B1, OATP1B3, OATP2B1) and to the Na+/taurocholate cotransporting polypeptide (NTCP) using stable transfected human embryonic kidney (HEK) 293 cells. In competition assays, Gd-EOB-DTPA inhibited the uptake of bromosulfophthalein (BSP) by OATP1B1 (IC50 = 0.6 mM) and OATP1B3 (IC50 = 0.4 mM). In comparison, the IC50 values for rifampicin were 11.9 (OATP1B1), 1.4 (OATP1B3), and 80.5 μM (OATP2B1), respectively. Uptake of BSP by OATP2B1, uptake of taurocholic acid by NTCP, and viability of all HEK cells were not influenced by Gd-EOB-DTPA in concentrations up to 10 mM. In uptake assays using a new liquid chromatography-tandem mass spectrometry method for quantification, Gd-EOB-DTPA was a substrate for OATP1B1 (Km = 0.7 mM, Vmax = 10.5 pmol/mg × min), OATP1B3 (Km = 4.1 mM, Vmax = 22.7 pmol/mg × min), and NTCP (Km = 0.04 mM, Vmax = 1.4 pmol/mg × min). The uptake by OATP2B1 was not different from the vector control. In conclusion, Gd-EOB-DTPA is a substrate of the liver-specific OATP1B1, OATP1B3, and NTCP.

Protein abundance of clinically relevant multidrug transporters along the entire length of the human intestine

Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

Visualization of Hepatic Uptake Transporter Function in Healthy Subjects by Using Gadoxetic Acid–enhanced MR Imaging

PURPOSE To determine if genetic polymorphisms of liver-specific human organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 influence cellular uptake of gadoxetic acid in vitro and if functionally relevant polymorphisms are confounders for liver enhancement by gadoxetic acid in healthy subjects. MATERIALS AND METHODS This study received ethics approval, and all subjects provided written informed consent. Cellular uptake of gadoxetic acid by OATP1B1 and OATP1B3 and their frequent genetic variants was measured by using stable transfected embryonic kidney HEK293 cells. Liver signal intensity at gadoxetic acid-enhanced MR imaging and pharmacokinetics of gadoxetic acid were evaluated in 36 healthy carriers of SLCO1B1/1B3 wild-type alleles (n = 10), SLCO1B1*1b/*1b (n = 8), SLCO1B1*15/*15 (n = 7), SLCO1B1*5/*15 (n = 1), SLCO1B1*1a/*5 (n = 6), and SLCO1B3*4/*4 (n = 4) by using T1-weighted MR imaging and liquid chromatography tandem mass spectrometry. RESULTS Transport activity for gadoxetic acid was increased in cells transfected with SLCO1B1c.388A>G (12.8 pmol/[mg·min]6 3.53, P = .001) but decreased in cells with SLCO1B1c.388A>G/521T>C (3.11 pmol/[mg·min] ± 0.918, P = .004) compared with cells with nonvariant transporter (6.32 pmol/[mg·min] ± 2.73). Compared with activity of cells transfected with the nonvariant SLCO1B3 (7.43 pmol/[mg·min] ± 2.43), SLCO1B3c.699G>A was a gain-of-function variant (15.1 pmol/[mg·min] ± 5.52, P = .002), whereas SLCO1B3c.334T>G (0.364 pmol/[mg·min] ± 0.125, P = .0001) and SLCO1B3c.1564G>T (0.295 pmol/[mg·min] ± 0.247, P = .0001) were variants with lower function. Liver enhancement with gadoxetic acid was reduced in subjects with OATP1B1*1a/*5 compared with wild-type subjects and those with OATP1B1*1b/*1b (area under enhancement curve, 3-480 minutes in arbitrary units [au]; 20.7 au ± 6.85 vs 36.5 au ± 8.08 [P = .006] vs 34.6 au ± 8.92 [P = .026]). The OATP1B3*4 polymorphism was not of functional relevance. No pharmacokinetic characteristics of gadoxetic acid were influenced by genetic polymorphisms of OATP1B1 and OATP1B3. CONCLUSION Liver-specific OATP1B1 and OATP1B3 are uptake carriers for gadoxetic acid in subjects. Genetic polymorphisms of OATP1B1 are signal confounders in gadoxetic acid-enhanced liver MR imaging.

Intestinal expression of P‐glycoprotein (ABCB1), multidrug resistance associated protein 2 (ABCC2), and uridine diphosphate–glucuronosyltransferase 1A1 predicts the disposition and modulates the effects of the cholesterol absorption inhibitor ezetimibe in humans

Ezetimibe is an inhibitor of the cholesterol uptake transporter Niemann‐Pick C1‐like protein (NPC1L1). Target concentrations can be influenced by intestinal uridine diphosphate–glucuronosyltransferases (UGTs) and the efflux transporters P‐glycoprotein (P‐gp) (ABCB1) and multidrug resistance associated protein 2 (MRP2) (ABCC2). This study evaluates the contribution of these factors to the disposition and cholesterol‐lowering effect of ezetimibe before and after induction of UGT1A1, P‐gp, and MRP2 with rifampin (INN, rifampicin).

Disposition of ezetimibe is influenced by polymorphisms of the hepatic uptake carrier OATP1B1

Objectives Genetic variability in hepatic uptake was recently shown to influence the disposition and cholesterol-lowering effects of statins. Ezetimibe, an inhibitor of the intestinal cholesterol uptake protein Niemann-Pick C 1 like 1, is another drug for which genetic polymorphisms of hepatic organic anion transporting polypeptides (OATPs) are expected to be of clinical relevance because ezetimibe undergoes intensive enterohepatic circulation for which hepatic uptake transporters may be rate-limiting determinants. Methods Using OATP1B3-, OATP2B1-, and OATP1B1-transfected HEK cells, including the OATP1B1 variants OATP1B1*1b and OATP1B1*5, we measured the uptake of ezetimibe and its glucuronide and we analyzed the competition with the common OATP-substrate bromosulfophthalein. Disposition and sterol-lowering effects of 20-mg ezetimibe were measured in 35 healthy participants genotyped for OATP1B1, ABCB1, ABCC2, and UGT1A1. Results Ezetimibe glucuronide inhibited bromosulfophthalein uptake in all OATP-transfected cells (50% inhibitory concentration (IC50): 0.14–0.26 μmol/l) whereas ezetimibe was 30–100 times less potent. Only the glucuronide was accumulated significantly in cells expressing OATP1B1 and OATP2B1. Its uptake in cells expressing OATP1B1*1b and *5 was reduced. In-vivo studies showed there was a gene-dose-dependent decrease in the area under the curve of ezetimibe in participants with the OATP1B1*1b protein (*1a/*1a, N=12, 112±66 ng×h/ml vs. *1a/*1b, N=8, 88±39 ng×h/ml vs. *1b/*1b, N=5, 55±18 ng×h/ml; Jonkheere–Terpstra, P=0.041) and a tendency for increased glucuronide exposure (704±296 vs. 878±369 vs. 1059±363 ng×h/ml; P=0.092). Fecal ezetimibe excretion was significantly decreased whereas renal glucuronide excretion was increased in carriers of *1b/*1b. Fecal excretion was also diminished in carriers of OATP1B1*5 and *15. The sterol-lowering effect of ezetimibe was not influenced by OATP1B1 polymorphisms. Conclusion Pharmacokinetics of ezetimibe is influenced by OATP1B1 polymorphisms in healthy participants after single dose administration.

LC–MS/MS-based quantification of clinically relevant intestinal uptake and efflux transporter proteins

Multidrug transporter proteins are crucial determinants in the pharmacokinetics of many drugs. To evaluate their impact on intestinal drug absorption, we developed and validated quantification methods for 10 uptake transporters (OATP1A2, OATP2B1, PEPT1, ASBT, OCT1, OCT3) and efflux transporters (ABCB1, ABCC2, ABCC3, ABCG2) that have been reported to be expressed and to be of clinical relevance in the human intestine. Quantification was performed by targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based quantification of proteospecific peptides after tryptic digestion using stable isotope labeled internal standard peptides. The chromatography of the respective peptides was performed by gradient elution using a reversed phase (C18) column (Kinetex(®), 100 × 3.0 mm, 2.6 μm) and 0.1% formic acid (FA) and acetonitrile with 0.1% FA as mobile phases at a flow rate of 0.5 ml/min. The MS/MS detection was done in the positive multiple reaction monitoring (MRM) mode by monitoring in each case three mass transitions for the transporter-derived peptides and the internal standard peptides. The assays were validated with respect to specificity, linearity (0.1-25 nM), within-day and between-day accuracy and precision as well as stability according to current bioanalytical guidelines. Finally, the developed methods were used to determine the transporter protein content in human intestinal tissue (jejunum and ileum). The methods were shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to measure transporter proteins in the human intestine.

Pharmaceutical excipients influence the function of human uptake transporting proteins.

Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-β-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na(+)/taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E(3)S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17β-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC(50) < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC(50) values of 0.05% (TA) and 0.14% (E(3)S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.

Disposition and sterol‐lowering effect of ezetimibe are influenced by single‐dose coadministration of rifampin, an inhibitor of multidrug transport proteins

The disposition and sterol‐lowering effect of ezetimibe are associated with long‐lasting enterosystemic circulation, which is initiated by secretion of ezetimibe and its glucuronide via intestinal P‐glycoprotein (P‐gp) (ABCB1) and the multidrug resistance‐associated protein 2 (MRP2) (ABCC2) into gut lumen. Hepatic uptake and secretion may contribute to recycling. To obtain deeper insight into the intestinal and hepatic processes, the disposition of ezetimibe was studied in the presence of rifampin (INN, rifampicin), a modulator of P‐gp, MRP2, and hepatic organic anion (uptake) transporting polypeptides (OATPs) (SLCOs).

Oral Absorption of Clarithromycin Is Nearly Abolished by Chronic Comedication of Rifampicin in Foals

The delivery of clarithromycin (CRL) to its site of action in bronchial/alveolar epithelial cells (EC), bronchial epithelial lining fluid (ELF), and bronchoalveolar lavage cells (BALC) may be influenced by CYP3A4 and the drug transporters, ATP-binding cassette (ABC) B1 and ABCC2 and organic anion-transporting polypeptides (OATPs), which can be modulated and/or up-regulated via the nuclear pregnane X receptor (PXR) by rifampicin (RIF). Therefore, we evaluated the disposition and pulmonary distribution of CLR (7.5 mg/kg b.i.d., 21 days) and expression of ABCB1, ABCC2, OATP1A2, and OATP2B1 in EC and BALC before and after comedication of RIF (10 mg/kg b.i.d., 11 days) in nine healthy foals (41–61 days, 115–159 kg) in which the genetic homology of drug transporters is close to that of their human analogs. After RIF comedication, relative bioavailability of CLR decreased by more than 90%. Concentrations in plasma (29.8 ± 26.3 versus 462 ± 368 ng/ml), ELF (0.69 ± 0.66 versus 9.49 ± 6.12 μg/ml), and BALC (10.2 ± 10.2 μg/ml 264 ± 375 μg/ml; all P < 0.05) were lowered drastically, whereas levels of the metabolite 14-hydroxyclarithromycin were not elevated despite higher 4β-hydroxycholesterol/cholesterol plasma concentration ratio, a surrogate for CYP3A4 induction. In the presence of CLR, ABCC2 and PXR mRNA contents were significantly and coordinately (r2 = 0.664, P < 0.001) reduced in BALC after RIF. In EC, mRNA expression of OATP1A2 increased but that of OATP2B1 decreased (both P < 0.05). RIF interrupts oral absorption and decreases CRL plasma levels below the minimal inhibitory concentration for eradication of Rhodococcus equi. Evidence that RIF influences the cellular uptake of CLR in bronchial cells and the PXR expression in BALC in the presence of high CLR concentrations exists.

Disposition and Sterol-Lowering Effect of Ezetimibe in Multidrug Resistance-Associated Protein 2-Deficient Rats

Disposition of the lipid-lowering agent ezetimibe (EZ) and its glucuronide (GLUC), which is mainly formed by UDP-glucuronosyltransferase (UGT) 1A1, is influenced by the intestinal efflux transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) 2. To evaluate the role of Mrp2 in overall disposition and pharmacodynamic effects of EZ, wild-type and Mrp2-deficient (TR-negative) Lewis.1W rats (eight males each) fed with a cholesterol-enriched diet were orally treated with 5 mg/kg EZ for 14 days. EZ and GLUC in serum, urine, and feces, and cholesterol, campesterol, and sitosterol in serum, were assayed using liquid chromatography (LC)-tandem mass spectrometry and LC-mass spectrometry methods, respectively. Gene expression of Bsep (bile salt exporting pump), multidrug resistance (Mdr) 1a, Mdr1b, Mrp2, Mrp3, Ntcp (sodium taurocholate co-transporting polypeptide), organic anion transporting polypeptides (Oatp) 1, 2, 4, and Ugt1a1 was quantified in several tissues using real-time reverse transcription-polymerase chain reaction. Mrp2 deficiency resulted in lower serum levels and fecal excretion of EZ (1.4 ± 0.4 versus 3.1 ± 1.1 ng/ml; 115 ± 48 versus 361 ± 102 μg/day, both p < 0.01), whereas serum concentrations of GLUC were manyfold increased compared with wild type (196 ± 76 versus 23 ± 25 ng/ml; p < 0.01), associated with elevated renal excretion and decreased intestinal clearance (7.8 ± 3.1 versus 0.4 ± 0.4 μg/day, p < 0.01; 0.3 ± 0.3 versus 15 ± 17 ml/min; p < 0.05). The sterol-lowering effect of EZ was reduced in correlation to EZ serum levels (cholesterol: r = 0.449, p = 0.093; campesterol: r = 0.717, p = 0.003; sitosterol: r = 0.507, p = 0.054), whereas GLUC was inversely correlated (r = -0.743, p = 0.002; r =-0.768, p = 0.001; r =-0.634, p = 0.011). Disposition of EZ may have been additionally influenced by hepatic P-gp, Mrp3, and Ugt1a1, which were expressed significantly higher in Mrp2-deficient rats. Mrp2 deficiency in rats is associated with decreased sterol-lowering effect of ezetimibe, obviously caused by lower intestinal clearance of the glucuronide and decreased enterosystemic and enterohepatic recycling of the parent ezetimibe to the intestinal Niemann-Pick C 1-like 1 sterol-uptake compartment.