Phase I and II metabolism and MRP2-mediated export of bosentan in a MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 quadruple-transfected cell line.

BACKGROUND AND PURPOSE: Hepatic uptake (e.g. by OATP1B1), phase I and II metabolism (e.g. by CYP3A4, UGT1A1) and subsequent biliary excretion (e.g. by MRP2) are key determinants for the pharmacokinetics of numerous drugs. However, stably transfected cell models for the simultaneous investigation of transport and phase I and II metabolism of drugs are lacking. EXPERIMENTAL APPROACH: A newly established quadruple-transfected MDCKII-OATP1B1-CYP3A4-UGT1A1-MRP2 cell line was used to investigate metabolism and transcellular transport of the endothelin receptor antagonist bosentan. KEY RESULTS: Intracellular accumulation of bosentan equivalents (i.e. parent compound and metabolites) was significantly lower in all cell lines expressing MRP2 compared to cell lines lacking this transporter (P < 0.001). Accordingly, considerably higher amounts of bosentan equivalents were detectable in the apical compartments of cell lines with MRP2 expression (P < 0.001). HPLC and LC-MS measurements revealed that mainly unchanged bosentan accumulated in intracellular and apical compartments. Furthermore, the phase I metabolites Ro 48-5033 and Ro 47-8634 were detected intracellularly in cell lines expressing CYP3A4. Additionally, a direct glucuronide of bosentan could be identified intracellularly in cell lines expressing UGT1A1 and in the apical compartments of cell lines expressing UGT1A1 and MRP2. CONCLUSIONS AND IMPLICATIONS: These in vitro data indicate that bosentan is a substrate of UGT1A1. Moreover, the efflux transporter MRP2 mediates export of bosentan and most likely also of bosentan glucuronide in the cell system. Taken together, cell lines simultaneously expressing transport proteins and metabolizing enzymes represent additional useful tools for the investigation of the interplay of transport and metabolism of drugs.

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells.

BACKGROUND AND PURPOSE: The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. EXPERIMENTAL APPROACH: Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. KEY RESULTS: Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. CONCLUSIONS AND IMPLICATIONS: Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition.

[ST segmental changes and arrhythmias in obstructive sleep apnea]. / ST Strecken Veränderungen und Rhythmusstörungen bei obstruktiver Schlafapnoe.

Patients with obstructive sleep apnea show a fall in arterial oxygen saturation during apneas. Whether this is causing myocardial ischemia and consecutively ST segment depressions in the electrocardiogram is not known. Therefore 15 consecutive patients (53 +/- 8 years, apnea index 45 +/- 28, minimal oxygen saturation 71 +/- 14%) with OSA were studied by Holter electrocardiogram and polysomnography. History and exercise testing gave no evidence of coronary heart disease. Three patients had ventricular arrhythmias Lown IVA and 10 had Lown I or III. Three patients showed unspecific negative T waves or ST segment elevations. In no patient significant ST segment depression was found. It is concluded that OSA does not lead to ischemic ST segment depression in the absence of coronary heart disease. The cause of ventricular arrhythmias in OSA seems not be related to myocardial ischemia.

[Pulmonary histiocytosis X]. / Pulmonale Histiozytosis X.

This is a report on 2 adult patients suffering from pulmonary histiocytosis X. The aetiology and the diagnostic and therapeutic approach are discussed on the basis of a review of the literature. This is a rare disease that is triggered by a virus and immunologically conditioned, the disposition being genetically transferred. It is characterised by cells known as histiocytosis X cells with typical X bodies and immunocytochemically identifiable S 100 antigen. It will usually be necessary to perform an open biopsy of the lung to determine the histology of histiocytosis X. Roentgenologically pathognomonic signs are in particular ring-shaped structures of up to 5 mm diameter with a marginal edge. Lung function analysis revealing hypoxaemia after stress and, less significantly, diffusion capacity and vital capacity, are also among the most sensitive data pointing to histiocytosis X. Indication for treating the patients, who usually do not display prominent signs and symptoms, should be discrete because spontaneous remissions occur very frequently. If the patients display relevant signs and symptoms, corticosteroid long-term treatment over 12 months with 0.5-1.0 mg/kg body weight per day is recommended employing a slowly and progressively reduced dosage schedule. Chemotherapeutic drugs or thymus extracts are administered in a few rare instances.