Stephanie Häusler

Bosentan Is a Substrate of Human OATP1B1 and OATP1B3: Inhibition of Hepatic Uptake as the Common Mechanism of Its Interactions with Cyclosporin A, Rifampicin, and Sildenafil

The elimination process of the endothelin receptor antagonist bosentan (Tracleer) in humans is entirely dependent on metabolism mediated by two cytochrome P450 (P450) enzymes, i.e., CYP3A4 and CYP2C9. Most interactions with concomitantly administered drugs can be rationalized in terms of inhibition of these P450 enzymes. The increased bosentan concentrations observed in the presence of cyclosporin A, rifampicin, or sildenafil, however, are incompatible with this paradigm and prompted the search for alternative mechanisms governing these interactions. In the present article, we identify bosentan and its active plasma metabolite, Ro 48-5033 (4-(2-hydroxy-1,1-dimethyl-ethyl)-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-[2,2′]bipyrimidinyl-4-yl]-benzenesulfonamide), as substrates of the human organic anion transporting polypeptides (OATP) OATP1B1 and OATP1B3. Bosentan uptake into Chinese hamster ovary cells expressing these OATP transporters was efficiently inhibited by cyclosporin A and rifampicin with IC50 values significantly below their effective plasma concentrations in humans. The phosphodiesterase-5 inhibitor sildenafil was also shown to interfere with OATP-mediated transport, however, at concentrations above those achieved in therapeutic use. Therefore, inhibition of bosentan hepatic uptake may represent an alternative/complementary mechanism to rationalize some of the pharmacokinetic interactions seen in therapeutic use. A similar picture has been drawn for drugs like pitavastatin and fexofenadine, drugs that are mainly excreted in unchanged form. Bosentan elimination, in contrast, is entirely dependent on metabolism. Therefore, the described interactions with rifampicin, cyclosporin A, and, to a lesser extent, sildenafil represent evidence that inhibition of hepatic uptake may become the rate-limiting step in the overall elimination process even for drugs whose elimination is entirely dependent on metabolism.

Transporters involved in the hepatic uptake of 99mTc-mebrofenin and indocyanine green

BACKGROUND & AIMS (99m)Tc-mebrofenin hepatobiliary scintigraphy (HBS) and the indocyanine green (ICG) clearance test are used for the assessment of hepatic function before and after liver surgery. The hepatic uptake of (99m)Tc-mebrofenin and ICG is considered similar to the uptake of organic anions such as bilirubin and bile acids. Little is known about hepatic uptake mechanisms of both compounds and recent evidence suggests that the hepatic transporters for ICG and (99m)Tc-mebrofenin are distinct. The aim of this study was to identify the specific human hepatic transporters of (99m)Tc-mebrofenin and ICG. METHODS The uptake of (99m)Tc-mebrofenin was investigated in cRNA-injected Xenopus laevis oocytes expressing human OATP1B1, OATP1B3, OATP2B1, or NTCP. Chinese hamster ovary (CHO) cells stably expressing OATP1B1, OATP1B3, OATP2B1, or NTCP were used as a mammalian expression system. ICG transport into CHO cells was additionally imaged with confocal microscopy. RESULTS We demonstrated that OATP1B1 and OATP1B3 are involved in the transport of (99m)Tc-mebrofenin. OATP1B1 showed an approximately 1.5-fold higher affinity for (99m)Tc-mebrofenin compared to OATP1B3. ICG is transported by OATP1B3 and NTCP. CONCLUSIONS The transporter specificity of (99m)Tc-mebrofenin and ICG partially overlaps as both compounds are transported by OATP1B3. (99m)Tc-mebrofenin is also taken up by OATP1B1, whereas ICG is additionally transported by NTCP.

Hepatic transport mechanisms of Cholyl-L-Lysyl-Fluorescein.

Cholyl-l-lysyl-fluorescein (CLF) is a fluorescent bile salt derivative that is being developed as an agent for determining in vivo liver function. However, the mechanisms of uptake and excretion by hepatocytes have not been rigorously studied. We have directly assessed the transport capacity of various hepatobiliary transporters for CLF. Uptake experiments were performed in Chinese hamster ovary cells transfected with human NTCP, OATP1B1, OATP1B3, and OATP2B1. Conversely, excretory systems were tested with plasma membrane vesicles from Sf21 insect cells expressing human ABCB11, ABCC2, ABCC3, and ABCG2. In addition, plasma clearance and biliary excretion of CLF were examined in wild-type, Abcc2(−/−), and Abcc3(−/−) mice. Human Na+-dependent taurocholic-cotransporting polypeptide (NTCP) and ATP-binding cassette B11 (ABCB11) were incapable of transporting CLF. In contrast, high-affinity transport of CLF was observed for organic anion-transporting polypeptide 1B3 (OATP1B3), ABCC2, and ABCC3 with Km values of 4.6 ± 2.7, 3.3 ± 2.0, and 3.7 ± 1.0 μM, respectively. In Abcc2(−/−) mice biliary excretion of CLF was strongly reduced compared with wild-type mice. This resulted in a much higher hepatic retention of CLF in Abcc2(−/−) versus wild-type mice: 64 versus 1% of the administered dose (2 h after administration). In mice intestinal uptake of CLF was negligible compared with that of taurocholate. Our conclusion is that human NTCP and ABCB11 are incapable of transporting CLF, whereas OATP1B3 and ABCC2/Abcc2 most likely mediate hepatic uptake and biliary excretion of CLF, respectively. CLF can be transported back into the blood by ABCC3. Enterohepatic circulation of CLF is minimal. This renders CLF suitable as an agent for assessing in vivo liver function.

Examining occupational self-efficacy, work locus of control and communication as moderators of the job insecurity-job performance relationship

Employees’ performance has been shown to be moderately hampered by job insecurity. Based on conservation of resources theory, the study examines whether three possible resources (occupational self-efficacy, work locus of control and communication) moderate the negative job insecurity—performance relationship. Analyses of a large Swiss dataset reveal two significant interaction effects: the higher the job insecurity, the less influence work locus of control and perceived communication exert on the job insecurity—performance relationship. This suggests that work locus of control and perceived communication may be resources that can only act beneficially in a situation of low job insecurity.

Vitamin D3 and Its Nuclear Receptor Increase the Expression and Activity of the Human Proton-Coupled Folate Transporter

Folates are essential for nucleic acid synthesis and are particularly required in rapidly proliferating tissues, such as intestinal epithelium and hemopoietic cells. Availability of dietary folates is determined by their absorption across the intestinal epithelium, mediated by the proton-coupled folate transporter (PCFT) at the apical enterocyte membranes. Whereas transport properties of PCFT are well characterized, regulation of PCFT gene expression remains less elucidated. We have studied the mechanisms that regulate PCFT promoter activity and expression in intestine-derived cells. PCFT mRNA levels are increased in Caco-2 cells treated with 1,25-dihydroxyvitamin D3 (vitamin D3) in a dose-dependent fashion, and the duodenal rat Pcft mRNA expression is induced by vitamin D3 ex vivo. The PCFT promoter region is transactivated by the vitamin D receptor (VDR) and its heterodimeric partner retinoid X receptor-α (RXRα) in the presence of vitamin D3. In silico analyses predicted a VDR response element (VDRE) in the PCFT promoter region −1694/−1680. DNA binding assays showed direct and specific binding of the VDR:RXRα heterodimer to the PCFT(−1694/−1680), and chromatin immunoprecipitations verified that this interaction occurs within living cells. Mutational promoter analyses confirmed that the PCFT(−1694/−1680) motif mediates a transcriptional response to vitamin D3. In functional support of this regulatory mechanism, treatment with vitamin D3 significantly increased the uptake of [3H]folic acid into Caco-2 cells at pH 5.5. In conclusion, vitamin D3 and VDR increase intestinal PCFT expression, resulting in enhanced cellular folate uptake. Pharmacological treatment of patients with vitamin D3 may have the added therapeutic benefit of enhancing the intestinal absorption of folates.

ABC-transporters are localized in caveolin-1-positive and reggie-1-negative and reggie-2-negative microdomains of the canalicular membrane in rat hepatocytes†

The canalicular plasma membrane is constantly exposed to bile acids acting as detergents. Bile acids are essential to mediate release of biliary lipids from the canalicular membrane. Membrane microdomains (previously called lipid rafts) are biochemically defined by their resistance to detergent solubilization at cold temperature. We aimed to investigate the canalicular plasma membrane for the presence of microdomains, which could protect this membrane against the detergent action of bile acids. Highly purified rat liver canalicular plasma membrane vesicles were extracted with 1% Triton X‐100 or 1% Lubrol WX at 4°C and subjected to flotation through sucrose step gradients. Both detergents yielded detergent‐resistant membranes containing the microdomain markers alkaline phosphatase and sphingomyelin. However, cholesterol was resistant to Lubrol WX solubilization, whereas it was only marginally resistant to solubilization by Triton X‐100. The microdomain marker caveolin‐1 was localized to the canalicular plasma membrane domain and was resistant to Lubrol WX, but to a large extent solubilized by Triton X‐100. The two additional microdomain markers, reggie‐1 and reggie‐2, were localized to the basolateral and canalicular plasma membrane and were partially resistant to Lubrol WX but resistant to Triton X‐100. The canalicular transporters bile salt export pump, multidrug resistance protein 2, multidrug resistance‐associated protein 2, and Abcg5 were largely resistant to Lubrol WX but were solubilized by Triton X‐100. Conclusion: These results indicate the presence of two different types of microdomains in the canalicular plasma membrane: “Lubrol‐microdomains” and “Triton‐microdomains”. “Lubrol‐microdomains” contain the machinery for canalicular bile formation and may be the starting place for canalicular lipid secretion. (HEPATOLOGY 2009.)

Effects of Farnesoid X Receptor Activation on Arachidonic Acid Metabolism, NF-kB Signaling, and Hepatic Inflammation

Inflammation has a recognized role in nonalcoholic fatty liver disease (NAFLD) progression. In the present work, we studied the effect of high-fat diet (HFD) on arachidonic acid metabolism in the liver and investigated the role of the farnesoid X receptor (FXR, NR1H4) in eicosanoid biosynthetic pathways and nuclear factor κ light-chain enhancer of activated B cells (NF-kB) signaling, major modulators of the inflammatory cascade. Mice were fed an HFD to induce NAFLD and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analyses were performed on liver tissue. Eicosanoid levels were measured from serum and urine samples. The molecular mechanism underlying the effect of FXR activation on arachidonic acid metabolism and NF-kB signaling was studied in human liver Huh7 cells and primary cultured hepatocytes. NAFLD was characterized by higher (∼25%) proinflammatory [leukotrienes (LTB4)] and lower (∼3-fold) anti-inflammatory [epoxyeicosatrienoic acids (EETs)] eicosanoid levels than in chow mice. OCA induced the expression of several hepatic cytochrome P450 (P450) epoxygenases, the enzymes responsible for EET synthesis, and mitigated HFD-induced hepatic injury. In vitro, induction of CYP450 epoxygenases was sufficient to inhibit NF-kB signaling and cell migration. The CYP450 epoxygenase pan-inhibitor gemfibrozil fully abolished the protective effect of OCA, indicating that OCA-mediated inhibition of NF-kB signaling was EET-dependent. In summary, NAFLD was characterized by an imbalance in arachidonate metabolism. FXR activation reprogramed arachidonate metabolism by inducing P450 epoxygenase expression and EET production. In vitro, FXR-mediated NF-kB inhibition required active P450 epoxygenases.

Fluorocholine transport mediated by the organic cation transporter 2 (OCT2,SLC22A2): implication for imaging of kidney tumors

[18F]fluorocholine is the fluorinated analog of [11C]choline and is used in positron emission tomography to monitor tumor metabolic activity. Although important to optimize its use and expand the clinical indications, the molecular determinants of fluorocholine cellular uptake are poorly characterized. In this work, we described the influx kinetics of fluorocholine mediated by the organic cation transporter 2 (OCT2, SLC22A2) and compared with that of choline. Then we characterized the expression pattern of OCT2 in renal cell carcinoma (RCC). In HEK293 cells stably transfected with OCT2 fluorocholine influx, kinetics was biphasic, suggesting two independent binding sites: a high-affinity (Km = 14 ± 8 µM, Vmax = 1.3 ± 0.5 nmol mg−1 min−1) and a low-affinity component (Km = 1.8 ± 0.3 mM, Vmax = 104 ± 4.5 nmol mg−1 min−1). Notably, choline was found to be transported with sigmoidal kinetics typical of homotropic positive cooperativity (h = 1.2, 95% confidence interval 1.1–1.3). OCT2 mRNA expression level was found significantly decreased in primary but not in metastatic RCC. Tissue microarray immunostaining of 216 RCC biopsies confirmed that the OCT2 protein level was consistent with that of the mRNA. The kinetic properties described in this work suggest that OCT2 is likely to play a dominant role in [18F]fluorocholine uptake in vivo. OCT2-altered expression in primary and metastatic cancer cells, as compared with the surrounding tissues, could be exploited in RCC imaging, especially to increase the detection sensitivity for small metastatic lesions, a major clinical challenge during the initial staging of RCC.