sincalide and estrone-sulfate

The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug-drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1. The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.

Topics: Atorvastatin; Biological Transport; Drug Interactions; Estradiol; Estrone; HEK293 Cells; Heptanoic Acids; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; In Vitro Techniques; Least-Squares Analysis; Liver; Liver-Specific Organic Anion Transporter 1; Models, Molecular; Multivariate Analysis; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Protein Isoforms; Pyrroles; Solute Carrier Organic Anion Transporter Family Member 1B3; Structure-Activity Relationship; Transfection

Cyclosporin A (CsA) causes a number of clinically relevant drug-drug interactions (DDIs) by inhibiting OATP1B1 and OATP1B3. In the present study, long-lasting inhibitory effects of CsA on these transporters were examined in comparison to tacrolimus (TCR). OATP1B1- and OATP1B3-expressing HEK293T cells, OATP1B1-expressing MDCK II cells, and human hepatocytes were preincubated with CsA or TCR, and uptake studies were carried out in their presence or absence. Western blot or immunohistochemical studies were done in OATP1B1-expressing HEK293T cells. The pretreatment of OATP1B1- and OATP1B3-expressing cells with 0.5-10 µM CsA, but not TCR, resulted in a reduction in their activity, even after washing out CsA from the incubation media. Preincubating the cells with CsA significantly enhanced its inhibitory effects on OATP1B1 and OATP1B3 by coincubation at 0.1-1 µM. Preincubation with 1 µM CsA caused a reduction in OATP1B1 activity for at least 18 h after its removal. The expression of OATP1B1 was not affected by incubation with CsA and no obvious change in its intracellular localization was observed. The long-lasting inhibition by CsA was also observed in human hepatocytes. Thus, CsA has a long-lasting inhibitory effect on OATP1B1 and OATP1B3. It may attribute to the clinically relevant DDIs between OATP substrates and CsA.

Topics: Animals; Blotting, Western; Cyclosporine; Dogs; Drug Interactions; Estrone; HEK293 Cells; Hepatocytes; Humans; Immunohistochemistry; Kinetics; Liver-Specific Organic Anion Transporter 1; Madin Darby Canine Kidney Cells; Models, Biological; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Sincalide; Solute Carrier Organic Anion Transporter Family Member 1B3; Tacrolimus; Transfection

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076bp, encoding a 692-amino acid protein with a molecular mass of approximately 85kDa. The Oatp1b4 gene is approximately 61kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine(2,5)]enkephalin (DPDPE), estradiol-17beta-glucuronide (E17betaG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17betaG and estrone-3-sulfate transports were monophasic with K(m) values of 5+/-1microM and 33+/-4microM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.

Topics: Amino Acid Sequence; Animals; Base Pairing; Base Sequence; Biological Transport; Cell Line; Cloning, Molecular; DNA, Complementary; Dogs; Enkephalin, D-Penicillamine (2,5)-; Estradiol; Estrone; Exons; Genes; Humans; Introns; Kidney; Kinetics; Liver; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Protein Structure, Tertiary; Sequence Homology, Amino Acid; Sincalide; Substrate Specificity; Sulfobromophthalein; Taurocholic Acid

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, V(max) and K(m), as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (P(dif)). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for V(max) and K(m) were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5-6-fold higher permeability at 37 degrees C compared with that at 4 degrees C), for fexofenadine a 16-fold higher passive permeability was seen at 37 degrees C. Therefore, P(dif) was better predicted if it was evaluated under the same experimental conditions as V(max) and K(m), i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37 degrees C, instead of a parallel control evaluation at 4 degrees C.

Topics: Algorithms; Animals; Biological Transport, Active; CHO Cells; Computer Simulation; Cricetinae; Cricetulus; Diffusion; Estrone; Fatty Acids, Monounsaturated; Fluvastatin; Hepatocytes; Indoles; Kinetics; Male; Membranes, Artificial; Models, Biological; Naphthalenes; Oligopeptides; Organic Anion Transporters; Permeability; Pharmaceutical Preparations; Pharmacokinetics; Piperidines; Quinolines; Rats; Rats, Wistar; Sincalide; Temperature; Terfenadine

Olmesartan, a novel angiotensin II AT1-receptor antagonist, is excreted into both bile and urine, with minimal metabolism. Because olmesartan is a hydrophilic anionic compound, some transporters could be involved in its hepatic and renal clearance. In this study, we characterized the role of human drug transporters in the pharmacokinetics of olmesartan and determined the contribution of each transporter to the overall clearance of olmesartan. Olmesartan was significantly taken up into human embryonic kidney 293 cells expressing organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, organic anion transporter (OAT) 1, and OAT3. We also observed its saturable uptake into human hepatocytes and kidney slices. Estimated from the relative activity factor method and application of specific inhibitors, the relative contributions of OATP1B1 and OATP1B3 to the uptake of olmesartan in human hepatocytes were almost the same, whereas OAT3 was predominantly involved in its uptake in kidney slices. The vectorial transport of olmesartan was observed in OATP1B1/multidrug resistance-associated protein (MRP) 2 double transfectants, but not in OATP1B1/multidrug resistance (MDR) 1 and OATP1B1/breast cancer resistance protein (BCRP) transfectants. ATP-dependent transport into membrane vesicles expressing human MRP2 and MRP4 was clearly observed, with K(m) values of 14.9 and 26.2 microM, respectively, whereas the urinary excretion of olmesartan in Mrp4-knockout mice was not different from that of control mice. We also investigated the transcellular transport of olmesartan medoxomil, a prodrug of olmesartan. Vectorial basal-to-apical transport was observed in OATP1B1/MRP2, OATP1B1/MDR1 double, and OATP1B1/BCRP double transfectants, suggesting the possible involvement of MRP2, MDR1, and BCRP in the limit of intestinal absorption of olmesartan medoxomil. From these results, we suggest that multiple transporters make a significant contribution to the pharmacokinetics of olmesartan and its prodrug.

Topics: Adenosine Triphosphate; Angiotensin II Type 1 Receptor Blockers; Animals; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cell Line; Dogs; Dose-Response Relationship, Drug; Estrone; Female; Hepatocytes; Humans; Imidazoles; In Vitro Techniques; Kidney; Kinetics; Liver; Liver-Specific Organic Anion Transporter 1; Membrane Transport Proteins; Mice; Mice, Knockout; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Olmesartan Medoxomil; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; p-Aminohippuric Acid; Penicillin G; Probenecid; Prodrugs; Protein Binding; Protein Isoforms; Sincalide; Solute Carrier Organic Anion Transporter Family Member 1B3; Tetrazoles; Transfection

Telmisartan, a nonpeptide angiotensin II receptor antagonist, is selectively distributed to liver. In the present study, we have characterized the contribution of organic anion transporting polypeptide (OATP) isoforms to the hepatic uptake of telmisartan by isolated rat hepatocytes, human cryopreserved hepatocytes, and human transporter-expressing cells. Because it is difficult to evaluate the transport activity of telmisartan because of its extensive adsorption to cells and culture materials, we performed the uptake study in the presence of human serum albumin. The saturable uptake of telmisartan into isolated rat hepatocytes took place in a Na(+)-independent manner and was inhibited by pravastatin, taurocholate, and digoxin, which are Oatp substrates and inhibitors, but not by organic cation, tetraethylammonium, indicating the involvement of Oatp isoforms in its uptake into rat hepatocytes. To identify which human OATP transporters are important for the hepatic uptake of telmisartan, the uptake assay was carried out using OATP1B1- and OATP1B3-expressing human embryonic kidney 293 cells and cryopreserved human hepatocytes. The uptake of telmisartan by OATP1B3-expressing cells was saturable (K(m) = 0.81 microM) and significantly higher than that by vector-transfected cells. In contrast, no significant uptake was observed in OATP1B1-expressing cells. We also observed the saturable uptake of telmisartan by human hepatocytes. Thirty micromolar estrone-3-sulfate, which can selectively inhibit OATP1B1-mediated uptake compared with OATP1B3, did not inhibit the uptake of telmisartan in human hepatocytes, whereas it could inhibit the uptake of estradiol 17beta-d-glucuronide mediated by OATP1B1. These results suggest that OATP1B3 is predominantly involved in the hepatic uptake of telmisartan in humans.

Topics: Angiotensin II Type 1 Receptor Blockers; Animals; Benzimidazoles; Benzoates; Cell Line; Estradiol; Estrone; Hepatocytes; Humans; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Rats; Rats, Sprague-Dawley; Serum Albumin; Sincalide; Solute Carrier Organic Anion Transporter Family Member 1B3; Telmisartan; Transfection

Valsartan is a highly selective angiotensin II AT1-receptor antagonist for the treatment of hypertension. Valsartan is mainly excreted into the bile in unchanged form. Because valsartan has an anionic carboxyl group, we hypothesized that a series of organic anion transporters could be involved in its hepatic clearance. In this study, to identify transporters that mediate the hepatic uptake and biliary excretion of valsartan and estimate the contribution of each transporter to the overall hepatic uptake and efflux, we characterized its transport using transporter-expressing systems, human cryopreserved hepatocytes, and Mrp2-deficient Eisai hyperbilirubinemic rats (EHBRs). Valsartan was significantly taken up into organic anion-transporting polypeptide (OATP) 1B1 (OATP2/OATP-C)- and OATP1B3 (OATP8)-expressing HEK293 cells. We also observed saturable uptake into human hepatocytes. Based on our estimation, the relative contribution of OATP1B1 to the uptake of valsartan in human hepatocytes depends on the batch, ranging from 20 to 70%. Regarding efflux transporters, the ratio of basal-to-apical transcellular transport of valsartan to that in the opposite direction in OATP1B1/MRP2 (multidrug resistance-associated protein 2) double transfected cells was the highest among the three kinds of double transfectants, OATP1B1/MRP2, OATP1B1/multi-drug resistance 1, and OATP1B1/breast cancer resistance protein-expressing MDCKII cells. We observed saturable ATP-dependent transport into membrane vesicles expressing human MRP2. We also found that the elimination of intravenously administered valsartan from plasma was markedly delayed, and the biliary excretion was severely impaired in EHBR compared with normal Sprague-Dawley rats. These results suggest that OATP1B1 and OATP1B3 as the uptake transporters and MRP2 as the efflux transporter are responsible for the efficient hepatobiliary transport of valsartan.

Topics: Adenosine Triphosphate; Angiotensin II Type 1 Receptor Blockers; Animals; Bile; Cell Line; Cell Membrane; Disease Models, Animal; Estrone; Hepatocytes; Humans; Hyperbilirubinemia; Liver-Specific Organic Anion Transporter 1; Male; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Rats; Rats, Sprague-Dawley; Sincalide; Solute Carrier Organic Anion Transporter Family Member 1B3; Tetrazoles; Transfection; Valine; Valsartan

Fexofenadine hydrochloride (FEX), a second generation H(1)-receptor antagonist, is mainly eliminated from the liver into bile in unchanged form. Recent studies have shown that FEX can be accepted by human MDR1 (P-glycoprotein), OATP1A2 [organic anion-transporting polypeptide (OATP)-A, and OATP2B1 (OATP-B)] expression systems. However, other transporters responsible for the hepatic uptake of FEX have not yet been identified. In the present study, we evaluated the contribution of OATP family transporters, namely OATP1B1 (OATP2/OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), to FEX uptake using transporter-expressing HEK293 (human embryonic kidney) cells. The uptake of FEX in OATP1B3-expressing cells was significantly greater than that in vector-transfected cells. On the other hand, OATP1B1- or OATP2B1-mediated uptake of FEX was not statistically significant. OATP1B3-mediated transport could be explained by a one-saturable component with a Michaelis constant (K(m)) of 108 +/- 11 microM. The inhibitory effect of FEX on the uptake of estrone-3-sulfate (E(1)S), cholecystokinin octapeptide (CCK-8), and 17beta-estradiol-17beta-d-glucuronide (E(2)17betaG) was also examined. Both OATP1B1- and OATP1B3-mediated E(2)17betaG uptake was inhibited by FEX. The K(i) values were 148 +/- 61 and 205 +/- 72 microM for OATP1B1 and OATP1B3, respectively. FEX also inhibited OATP1B3-mediated CCK-8 uptake and OATP1B1-mediated E(1)S uptake with a K(i) value of 83.3 +/- 15.3 and 257 +/- 84 microM, respectively, suggesting that FEX could not be used as a specific inhibitor for OATP1B1 and OATP1B3, although FEX was preferentially accepted by OATP1B3. In conclusion, this is, to our knowledge, the first demonstration that OATP1B3 is thought to be a major transporter involved in hepatic uptake of FEX in humans.

Topics: Cell Line; Estradiol; Estrone; Histamine H1 Antagonists; Humans; Liver; Liver-Specific Organic Anion Transporter 1; Organic Anion Transport Protein 1; Sincalide; Terfenadine

Pitavastatin, a novel potent 3-hydroxymethylglutaryl-CoA reductase inhibitor, is selectively distributed to the liver in rats. However, the hepatic uptake mechanism of pitavastatin has not been clarified yet. In the present study, we investigated the contribution of organic anion transporting polypeptide 2 (OATP2/OATP1B1) and OATP8 (OATP1B3) to pitavastatin uptake using transporter-expressing HEK293 cells and human cryopreserved hepatocytes. Uptake studies using OATP2- and OATP8-expressing cells revealed a saturable and Na(+)-independent uptake, with K(m) values of 3.0 and 3.3 microM for OATP2 and OATP8, respectively. To determine which transporter is more important for its hepatic uptake, we proposed a methodology for estimating their quantitative contribution to the overall hepatic uptake by comparing the uptake clearance of pitavastatin with that of reference compounds (a selective substrate for OATP2 (estrone-3-sulfate) and OATP8 (cholecystokinin octapeptide) in expression systems and human hepatocytes. The concept of this method is similar to the so-called relative activity factor method often used in estimating the contribution of each cytochrome P450 isoform to the overall metabolism. Applying this method to pitavastatin, the observed uptake clearance in human hepatocytes could be almost completely accounted for by OATP2 and OATP8, and about 90% of the total hepatic clearance could be accounted for by OATP2. This result was also supported by estimating the relative expression level of each transporter in expression systems and hepatocytes by Western blot analysis. These results suggest that OATP2 is the most important transporter for the hepatic uptake of pitavastatin in humans.

Topics: Biological Transport; Blotting, Western; Cells, Cultured; Drug Interactions; Estrone; Hepatocytes; Humans; Liver; Liver-Specific Organic Anion Transporter 1; Organic Anion Transporters, Sodium-Independent; Quinolines; Sincalide; Sodium; Solute Carrier Organic Anion Transporter Family Member 1B3

The objective of this study was to examine the transport activity of the human organic anion transporter OATP-C (SLC21A6) for oligopeptides that are eliminated rapidly from the systemic circulation. We focused on an opioid peptide analogue, [D-Ala(2), D-Leu(5)]-enkephalin (DADLE), a linear pentapeptide modified to be stable. [(3)H]DADLE was taken up by rat isolated hepatocytes in a saturable manner and highly accumulated in the liver after intravenous administration to rats. The uptake of [(3)H]DADLE by the isolated hepatocytes was inhibited by several organic anions and pentapeptides, but not by tetra- or tripeptides. When OATP-C was expressed in Xenopus laevis oocytes, a significant increase in uptake of [(3)H]DADLE was observed. Moreover, the inhibitory effects of various compounds, including some peptides, on [(3)H]estrone-3-sulfate uptake by OATP-C were similar to those observed in [(3)H]DADLE uptake by rat isolated hepatocytes. In conclusion, it was demonstrated that OATP-C contributes to the rapid hepatic excretion of peptides and peptide-mimetic drugs.

Topics: Animals; Anions; Bile; Biological Transport; Enkephalin, D-Penicillamine (2,5)-; Enkephalin, Leucine-2-Alanine; Estrone; Hepatocytes; In Vitro Techniques; Injections, Intravenous; Liver-Specific Organic Anion Transporter 1; Oocytes; Peptides; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Xenopus laevis