digoxin and estradiol-17-beta-glucuronide

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

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β-D-glucuronide (E(2)-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly (p<0.01) decreased the accumulation of estrone-3-sulfate and E(2)-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant (p<0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R(2)=0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Topics: 1-Methyl-4-phenylpyridinium; Adolescent; Adult; Biological Transport; Corticosterone; Cryopreservation; Digoxin; Estradiol; Estrone; Hepatocytes; Humans; Male; Membrane Transport Proteins; Middle Aged; Organic Anion Transporters; Organic Anion Transporters, Sodium-Dependent; Organic Anion Transporters, Sodium-Independent; Organic Cation Transport Proteins; Organic Cation Transporter 1; Prazosin; Rifampin; RNA, Messenger; Solute Carrier Organic Anion Transporter Family Member 1B3; Symporters; Taurocholic Acid

The chemical space of registered oral drugs was explored for inhibitors of the human multidrug-resistance associated protein 2 (MRP2; ABCC2), using a data set of 191 structurally diverse drugs and drug-like compounds. The data set included a new reference set of 75 compounds, for studies of hepatic drug interactions with transport proteins, CYP enzymes, and compounds associated with liver toxicity. The inhibition of MRP2-mediated transport of estradiol-17beta-D-glucuronide was studied in inverted membrane vesicles from Sf9 cells overexpressing human MRP2. A total of 27 previously unknown MRP2 inhibitors were identified, and the results indicate an overlapping but narrower inhibitor space for MRP2 compared with the two other major ABC efflux transporters P-gp (ABCB1) and BCRP (ABCG2). In addition, 13 compounds were shown to stimulate the transport of estradiol-17beta-D-glucuronide. The experimental results were used to develop a computational model able to discriminate inhibitors from noninhibitors according to their molecular structure, resulting in a predictive power of 86% for the training set and 72% for the test set. The inhibitors were in general larger and more lipophilic and presented a higher aromaticity than the noninhibitors. The developed computational model is applicable in an early stage of the drug discovery process and is proposed as a tool for prediction of MRP2-mediated hepatic drug interactions and toxicity.

Topics: Administration, Oral; Animals; Antineoplastic Agents; Antipsychotic Agents; Antiviral Agents; 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; Biological Transport; Cell Line; Computer Simulation; Cytochrome P-450 Enzyme System; Drug-Related Side Effects and Adverse Reactions; Estradiol; Humans; Insecta; Liver; Models, Molecular; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Neoplasm Proteins; Pharmaceutical Preparations; Pharmacology; Structure-Activity Relationship

Fresh hepatocytes cultured in a sandwich configuration allow for the development of intact bile canaliculi and the ability to measure hepatic uptake and biliary clearance. A disadvantage of this model is its dependence upon hepatocytes from fresh tissue. Therefore, the ability to use cryopreserved human hepatocytes in this model would be a great advantage. Multiple variables were tested, and the recommended conditions for culturing cryopreserved human hepatocytes in a sandwich configuration in 24-well plates are as follows: BioCoat plates, a cell density of 0.35 x 10(6) cells/well in 500 microl, an overlay of Matrigel and InVitroGRO media. These conditions resulted in good hepatocyte morphology and the formation of distinct bile canaliculi. The function of multiple uptake and efflux transporters was tested in multiple lots of cryopreserved and fresh human hepatocytes. For taurocholate [Na+ taurocholate cotransporting polypeptide/organic anion transporting polypeptide (OATP) uptake/bile salt export pump efflux], the average apparent uptake, apparent intrinsic biliary clearance, and biliary excretion index among five cryopreserved hepatocyte lots was high, ranging from 11 to 17 pmol/min/mg protein, 5.8 to 10 microl/min/mg protein, and 41 to 63%, respectively. The corresponding values for digoxin (OATP-8 uptake/multidrug resistance protein 1 efflux) were 0.69 to 1.5 pmol/min/mg protein, 0.60 to 1.5 microl/min/mg protein, and 37 to 63%. Both substrates exhibited similar results when fresh human hepatocytes were used. In addition, substrates of breast cancer resistance protein and multidrug resistance-associated protein 2 were also tested in this model, and all cryopreserved lots showed functional transport of these substrates. The use of cryopreserved human hepatocytes in 24-well sandwich culture to form intact bile canaliculi and to exhibit functional uptake and efflux transport has been successfully demonstrated.

Topics: Adolescent; Aged; Bile Canaliculi; Biological Transport; Cell Culture Techniques; Cells, Cultured; Child, Preschool; Cryopreservation; Digoxin; Drug Evaluation, Preclinical; Estradiol; Female; Hepatocytes; Humans; Kinetics; Male; Membrane Transport Proteins; Middle Aged; Taurocholic Acid

Rat organic anion transporting polypeptide 2 (rOatp2) is a member of the OATP family. It exhibits broad substrate specificity and accepts amphipathic organic anions, cardiac glycosides (digoxin and ouabain; a neutral compound), and organic cations (rocuronium and N-(4,4-azo-n-pentyl)-21-deoxyajamalinium). In the present study, kinetic analyses were carried out to investigate whether taurocholate (TCA), digoxin, and 17beta-estradiol-D-17beta-glucuronide (E(2)17betaG) share the same recognition site on rOatp2 for their transport. The transport of TCA and digoxin was mutually inhibited, and the K(i) values of digoxin and TCA for the transport of TCA and digoxin were 0.58 and 160 microM, respectively. The K(m) and V(max) values of TCA and digoxin were 190 microM and 140 pmol/min/mg of protein and 1.1 microM and 6.6 pmol/min/mg of protein, respectively. The K(m) and K(i) values were consistent. In addition, digoxin (1 microM) and TCA (100 microM) increased the K(m) values of TCA and digoxin, respectively, but they did not affect the V(max) values, suggesting that their inhibition is competitive. The transport of digoxin via rOatp2 was inhibited slightly by E(2)17betaG, whereas the uptake of TCA was stimulated by E(2)17betaG in a concentration-dependent manner. These results suggest that rOatp2 has at least two substrate recognition sites, one for TCA and digoxin and the other for E(2)17betaG.

Topics: Animals; Detergents; Digoxin; Enzyme Inhibitors; Estradiol; LLC-PK1 Cells; Organic Anion Transporters, Sodium-Independent; Rats; Substrate Specificity; Swine; Taurocholic Acid

Transport of various amphipathic organic compounds is mediated by organic anion transporting polypeptides (OATPs in humans, Oatps in rodents), which belong to the solute carrier family 21A (SLC21A/Slc21a). Several of these transporters exhibit a broad and overlapping substrate specificity and are expressed in a variety of different tissues. We have isolated and functionally characterized OATP-F (SLC21A14), a novel member of the OATP family. The cDNA (3059 bp) contains an open reading frame of 2136 bp encoding a protein of 712 amino acids. Its gene containing 15 exons is located on chromosome 12p12. OATP-F exhibits 47-48% amino acid identity with OATP-A, OATP-C, and OATP8, the genes of which are clustered on chromosome 12p12. OATP-F is predominantly expressed in multiple brain regions and Leydig cells of the testis. OATP-F mediates high affinity transport of T(4) and reverse T(3) with apparent K(m) values of approximately 90 nM and 128 nM, respectively. Substrates less well transported by OATP-F include T(3), bromosulfophthalein, estrone-3-sulfate, and estradiol-17beta-glucuronide. Furthermore, OATP-F-mediated T(4) uptake could be cis-inhibited by L-T(4) and D-T(4), but not by 3,5-diiodothyronine, indicating that T(4) transport is not stereospecific, but that 3',5'-iodination is important for efficient transport by OATP-F. Thus, in contrast to most other family members, OATP-F has a more selective substrate preference and may play an important role in the disposition of thyroid hormones in brain and testis.

Topics: Amino Acid Sequence; Animals; Brain; CHO Cells; Chromosomes, Human, Pair 12; Cloning, Molecular; Cricetinae; Diiodothyronines; Estradiol; Estrone; Female; Humans; Leydig Cells; Male; Membrane Proteins; Molecular Sequence Data; Oocytes; Organ Specificity; Organic Anion Transporters; Sequence Homology, Amino Acid; Sulfobromophthalein; Testis; Thyroxine; Triiodothyronine; Xenopus

P-glycoprotein (P-gp) and organic anion transporting polypeptides (Oatp) are expressed at the blood-brain barrier (BBB). There is little functional evidence for Oatp-mediated transport at the BBB. The peptidic delta opioid-receptor agonist [D-penicillamine(2,5)]-enkephalin (DPDPE) is a substrate of mdr1a P-gp and Oatp2. The present study evaluated the influence of these transporters on brain uptake of DPDPE by in situ perfusion in mice. Brain uptake was increased approximately 12-fold in mice lacking P-gp in the BBB, but the P-gp inhibitor dexverapamil did not increase uptake in P-gp-competent mice. In P-gp-deficient mice, DPDPE uptake was saturable (K(m) approximately 24 mM), and was inhibited by dexverapamil and the Oatp2 substrates digoxin, estradiol-17beta-glucuronide and fexofenadine. These results confirm P-gp-mediated efflux of DPDPE, and suggest functional uptake transport of DPDPE by Oatp, at the murine BBB.

Topics: Analgesics, Opioid; Animals; Anion Transport Proteins; ATP Binding Cassette Transporter, Subfamily B; ATP-Binding Cassette Transporters; Blood-Brain Barrier; Calcium Channel Blockers; Carrier Proteins; Digoxin; Enkephalin, D-Penicillamine (2,5)-; Enzyme Inhibitors; Estradiol; Histamine H1 Antagonists; Male; Mice; Mice, Knockout; Perfusion; Receptors, Opioid, delta; Terfenadine; Verapamil