rifampin and estrone-sulfate

We evaluated the effects of cytochrome P450 3A4 (CYP3A4) induction and inhibition on steady-state pharmacokinetics of the components of a novel oral contraceptive (OC) containing estradiol valerate (E₂V) and dienogest (DNG).. CYP3A4 induction was assessed in an open-label, one-arm study. Sixteen healthy postmenopausal women received E₂V 2 mg/DNG 3 mg (days 1-17) and concomitant rifampicin (600 mg, days 12-16). Ratios of the area under the serum concentration-time curve between 0 and 24 h [AUC(0-24 h)] and maximum serum concentration (C(max)) of E₂ and DNG on days 17 and 11 (after and before rifampicin intervention) are presented. CYP3A4 inhibition was investigated in an open-label, parallel-group study in 24 healthy postmenopausal women receiving E₂V 2 mg/DNG 3 mg (days 1-14) and concomitant ketoconazole (400 mg, n=12) or erythromycin (500 mg three times daily, n=12) on days 8-14. Mean ratios of AUC(0-24 h) and C(max) of E₂ and DNG on days 7 and 14 are presented.. Concomitant administration of rifampicin decreased systemic drug exposure and yielded geometric mean ratios for E₂C(max) and AUC(0-24 h) of 75% and 56%, respectively. Corresponding mean ratios for DNG were 48% and 17%, respectively. Ketoconazole coadministration increased systemic drug exposure and yielded ratios of E₂ of 165% and 157%, respectively, and ratios of DNG of 194% and 286%, respectively. Erythromycin coadministration also resulted in increased mean C(max) and AUC(0-24 h) of both E₂ and DNG. Geometric mean ratios of C(max) and AUC(0-24 h) for E₂ were 151% and 133%, respectively. Corresponding ratios for DNG were 133% and 162%, respectively.. Significant drug-drug interactions are apparent when CYP3A4 modulators are coadministered with the components of a novel OC containing E₂V/DNG. Coadministration of CYP3A4 modulators should be avoided where possible, and another type of contraception should be used when coadministration of CYP3A4 inducers like rifampicin is unavoidable.

Topics: Aged; Anti-Infective Agents; Biotransformation; Contraceptives, Oral, Combined; Contraceptives, Oral, Hormonal; Cross-Over Studies; Cytochrome P-450 CYP3A; Drug Combinations; Drug Interactions; Enzyme Induction; Erythromycin; Estradiol; Estrogen Replacement Therapy; Estrone; Female; Humans; Ketoconazole; Middle Aged; Nandrolone; Postmenopause; Rifampin

Human hepatocytes that had been cold-preserved in SureTran(TM) matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012). After 5 d of cold preservation, the viability was still more than 70%, and after 8 d it was around 60%. In hepatocytes that had been cold-preserved for 3 d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8 µM rifampicin for 72 h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3 d and thereafter treated with 1 mM phenobarbital for 72 h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3 d, while the activity increased in cells treated with 0.3-25 µM β-naphthoflavone for 72 h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2 d. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.

Topics: Cell Culture Techniques; Cell Survival; Cells, Cultured; Cytochrome P-450 CYP3A; Estrone; Fluorobenzenes; Hepatocytes; Humans; Ipratropium; Liver; Organic Anion Transporters; Phenobarbital; Pyrimidines; Rifampin; RNA, Messenger; Rosuvastatin Calcium; Sulfonamides; Taurocholic Acid

Organic anion transporting polypeptides (OATP) have been extensively recognized as key determinants of absorption, distribution, metabolism, and excretion of various drugs because of their broad substrate specificity, wide tissue distribution, and the involvement of drug-drug interaction. As the first cloned human OATP, OATP1A2 has been found to transport a wide spectrum of endogenous and exogenous compounds. Bovine Oapt1a2 shared high homology with the human transporter and is considered as its functional ortholog. In the present study, we expressed bovine Oatp1a2 in human embryonic kidney 293 cells and found that, unlike human OATP1A2, the transport of estrone-3-sulfate (E-3-S) exhibited biphasic saturation kinetics. The K(m) values are 0.25 ± 0.08 and 46.6 ± 18.5 µM, and V(max) values were 24.5 ±4.4 and 375 ± 142 pmol/mg protein/min for high- and low-affinity sites, respectively, suggesting the presence of multiple binding sites. Further study on other Oatp1a2 substrates showed that the high affinity component for E-3-S is responsible for the interaction with taurocholate, bromsulphthalein, and rifampicin and is sensitive to proton concentration change, whereas the low affinity binding site is only involved in the binding of the antitumor drug methotrexate and had no response to change of pH.

Topics: Amino Acid Sequence; Animals; Binding Sites; Binding, Competitive; Cattle; Estrone; HEK293 Cells; Humans; Hydrogen-Ion Concentration; Kinetics; Methotrexate; Organic Anion Transporters; Protein Structure, Secondary; Recombinant Proteins; Rifampin; Sulfobromophthalein; Taurocholic Acid; Transfection

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 ligand-activated nuclear receptor pregnane X receptor (PXR) is known to play a role in the regulated expression of drug metabolizing enzymes and transporters. Recent studies suggest a potential clinically relevant role of PXR in breast cancer. However, the relevant pathway or target genes of PXR in breast cancer biology and progression have not yet been fully clarified. In this study, we show that mRNA expression of organic anion transporter polypeptide 1A2 (OATP1A2), a transporter capable of mediating the cellular uptake of estrogen metabolites, is nearly 10-fold greater in breast cancer compared with adjacent healthy breast tissues. Immunohistochemistry revealed exclusive expression of OATP1A2 in breast cancer tissue. Interestingly, treatment of breast cancer cells in vitro with the PXR agonist rifampin induced OATP1A2 expression in a time-dependent and concentration-dependent manner. Consistent with its role as a hormone uptake transporter, induction of OATP1A2 was associated with increased uptake of estrone 3-sulfate. The rifampin response was abrogated after small interfering RNA targeting of PXR. We then identified a PXR response element in the human OATP1A2 promoter, located approximately 5.7 kb upstream of the transcription initiation site. The specificity of PXR-OATP1A2 promoter interaction was confirmed using chromatin immunoprecipitation. Importantly, we used a novel potent and specific antagonist of PXR (A-792611) to show the reversal of the rifampin effect on the cellular uptake of E(1)S. These data provide important new insights into the interplay between a xenobiotic nuclear receptor PXR and OATP1A2 that could contribute to the pathogenesis of breast cancer and may also prove to be heretofore unrecognized targets for breast cancer treatment.

Topics: Breast; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Chromatin Immunoprecipitation; Constitutive Androstane Receptor; Dipeptides; Estrogens; Estrone; Female; Humans; Neoplasm Staging; Organic Anion Transporters; Pregnane X Receptor; Promoter Regions, Genetic; Pyridines; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Receptors, Steroid; Rifampin; RNA, Messenger; Transcription Factors

The pregnane X receptor is a ligand-activated transcription factor that is abundantly expressed in hepatocytes. Numerous drugs are pregnane X receptor ligands. To bind to their receptor they must cross the sinusoidal membrane. Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are polyspecific transporters expressed at the sinusoidal membrane of human hepatocytes. They mediate transport of a variety of drugs including the pregnane X receptor ligands rifampicin and dexamethasone. To test whether additional pregnane X receptor ligands interact with OATP1B1- and 1B3-mediated transport, we developed Chinese Hamster Ovary (CHO) cell lines stably expressing OATP1B1 or 1B3 at high levels. OATP1B1- and 1B3-mediated estradiol-17beta-glucuronide uptake was inhibited by several pregnane X receptor ligands in a concentration dependent way. IC(50) values for rifampicin, paclitaxel, mifepristone, and troglitazone were within their respective pharmacological free plasma concentrations. Kinetic analysis revealed that clotrimazole inhibits OATP1B1-mediated estradiol-17beta-glucuronide transport with a K(i) of 7.7+/-0.3 microM in a competitive way. However, uptake of OATP1B3-mediated estradiol-17beta-glucuronide was stimulated and this stimulation was due to an increased apparent affinity. Transport of estrone-3-sulfate was hardly affected while all other substrates tested were inhibited. Additional azoles like fluconazole, ketoconazole and miconazole did not stimulate OATP1B3-mediated estradiol-17beta-glucuronide transport. In summary, these results demonstrate that pregnane X receptor ligands, by inhibiting or stimulating OATP-mediated uptake, can lead to drug-drug interactions at the transporter level.

Topics: Animals; CHO Cells; Chromans; Clotrimazole; Cricetinae; Cricetulus; Dose-Response Relationship, Drug; Drug Interactions; Estradiol; Estrone; Humans; Kinetics; Ligands; Liver-Specific Organic Anion Transporter 1; Mifepristone; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Paclitaxel; Pregnane X Receptor; Receptors, Steroid; Rifampin; Solute Carrier Organic Anion Transporter Family Member 1B3; Thiazolidinediones; Transfection; Troglitazone; Xenobiotics

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 IC(50) 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.

Topics: Animals; Aryl Hydrocarbon Hydroxylases; Biological Transport; Bosentan; CHO Cells; Cricetinae; Cricetulus; Cyclosporine; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dehydroepiandrosterone Sulfate; Drug Interactions; Enzyme Inhibitors; Estradiol; Estrone; Humans; Liver-Specific Organic Anion Transporter 1; Molecular Structure; Organic Anion Transporters; Organic Anion Transporters, Sodium-Independent; Piperazines; Purines; Pyrimidines; Rifampin; Sildenafil Citrate; Solute Carrier Organic Anion Transporter Family Member 1B3; Sulfonamides; Sulfones; Warfarin

The transcellular transport of many compounds, which cannot readily cross the lipid bilayer, is mediated by drug uptake and efflux transporters. Human OATP1B1 and MRP2 have been implicated in the hepato-biliary transport of many endogenous and exogenous compounds. Here, we have established epithelial porcine kidney LLC-PK1 derived cell lines, that express both transporters in a polarized fashion, as a model to predict hepato-biliary transport. Immunological identification of OATP1B1 in the recombinant cell lines was greatly facilitated by its C-terminal tagging with a peptide sequence derived from hemagglutinin (HA) avoiding the generation of OATP1B1 specific antibodies. Importantly, the tag did not interfere with the functionality of the transporter. Compared to LLC-PK1 cells and cells which expressed only OATP1B1, the cell line that co-expressed MRP2 and OATP1B1 displayed high directional basolateral-to-apical transport of 17 beta-estradiol-17 beta-glucuronide and estrone-3-sulfate. Dehydroepiandrosterone sulfate already displayed a significant basolateral-to-apical transport in the parental cell line, which was further stimulated upon expression of both transporters. Transcellular flux of all steroid conjugates in the opposite direction (apical-to-basolateral) was much lower. By employing this cellular model we were able to demonstrate for the first time that OATP1B1 together with MRP2 mediates the trans-cellular transport of rifampicin. It is anticipated that the models established herein will greatly facilitate the identification of transporters involved in the disposition of novel drug candidates.

Topics: Animals; Biological Transport; Epithelium; Estradiol; Estrone; Liver-Specific Organic Anion Transporter 1; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Rifampin; Swine; Transfection

Plasma estrone, estradiol, estrone sulfate, androstenedione, testosterone and sex hormone binding globulin were measured in 14 patients (3 postmenopausal women, 11 men) with tuberculosis who received rifampicin. During treatment a moderate, but significant increase in the plasma level of estradiol (mean increase 32%, P less than 0.01) and estrone (mean increase 13%, P less than 0.01) were seen. In contrast, plasma estrone sulfate was significantly reduced (mean reduction of 25%, P less than 0.05). No alteration in plasma testosterone was observed, but there was a slight (mean 15%) increase in plasma androstenedione of borderline significance (P = 0.052). In eight patients, from whom all tuberculostatic treatment except rifampicin had been withdrawn, plasma sex hormone binding globulin was found to be increased by 75% by rifampicin treatment. Further, the results obtained in this part of the study confirmed the alteration in plasma estrone sulfate to be caused by rifampicin alone without any contribution from other tuberculostatic drugs. While plasma estradiol could be increased due to elevation of sex hormone binding globulin, plasma estrone was probably increased secondary to the increase in plasma androstenedione. A reduced plasma estrone sulfate level suggests that rifampicin enhances the rate of estrone sulfate metabolism. The possibility that treatment with drugs which reduce plasma estrone sulfate might be beneficial for hormone dependent cancers is discussed.

Topics: Adult; Aged; Aged, 80 and over; Estradiol; Estrogens; Estrone; Female; Humans; Male; Middle Aged; Rifampin; Sex Hormone-Binding Globulin