Digoxin net secretory transport in bronchial epithelial cell layers is not exclusively mediated by P-glycoprotein/MDR1

Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are creditedThe impact of P-glycoprotein (MDR1, ABCB1) on drug disposition in the lungs as well as its presence and activity in in vitro respiratory drug absorption models remain controversial to date. Hence, we characterised MDR1 expression and the bidirectional transport of the common MDR1 probe 3H-digoxin in air-liquid interfaced (ALI) layers of normal human bronchial epithelial (NHBE) cells and of the Calu-3 bronchial epithelial cell line at different passage numbers. Madin-Darby Canine Kidney (MDCKII) cells transfected with the human MDR1 were used as positive controls. 3H-digoxin efflux ratio (ER) was low and highly variable in NHBE layers. In contrast, ER=11.4 or 3.0 was measured in Calu-3 layers at a low or high passage number, respectively. These were, however, in contradiction with increased MDR1 protein levels observed upon passaging. Furthermore, ATP depletion and the two MDR1 inhibitory antibodies MRK16 and UIC2 had no or only a marginal impact on 3H-digoxin net secretory transport in the cell line. Our data do not support an exclusive role of MDR1 in 3H-digoxin apparent efflux in ALI Calu-3 layers and suggest the participation of an ATP-independent carrier. Identification of this transporter might provide a better understanding of drug distribution in the lungs.Peer reviewe

Linking in vitro lipolysis and microsomal metabolism for the quantitative prediction of oral bioavailability of BCS II drugs administered in lipidic formulations

Lipidic formulations (LFs) are increasingly utilized for the delivery of drugs that belong to class II of the Biopharmaceutics Classification System (BCS). The current work proposes, for the first time, the combination of in vitro lipolysis and microsomal metabolism studies for the quantitative prediction of human oral bioavailability of BCS II drugs administered in LFs. Marinol® and Neoral® were selected as model LFs and their observed oral bioavailabilities (Fobserved) obtained from published clinical studies in humans. Two separate lipolysis buffers, differing in the level of surfactant concentrations, were used for digestion of the LFs. The predicted fraction absorbed (Fabs) was calculated by measuring the drug concentration in the micellar phase after completion of the lipolysis process. To determine first-pass metabolism (Fg∙Fh), drug depletion studies with human microsomes were performed. Clearance values were determined by applying the “in vitro half-life approach”. The estimated Fabs and Fg∙Fh values were combined for the calculation of the predicted oral bioavailability (Fpredicted). Results showed that there was a strong correlation between Fobserved and Fpredicted values only when Fabs was calculated using a buffer with surfactant concentrations closer to physiological conditions. The general accuracy of the predicted values suggests that the novel in vitro lipolysis/metabolism approach could quantitatively predict the oral bioavailability of lipophilic drugs administered in LFs

Chain length affects pancreatic lipase activity and the extent and pH–time profile of triglyceride lipolysis

Triglycerides (TG) are one of the most common excipients used in oral lipid-based formulations. The chain length of the TG plays an important role in the oral bioavailability of the co-administered drug. Fatty acid (FA) chain-length specificity of porcine pancreatic lipase was studied by means of an in vitro lipolysis model under bio-relevant conditions at pH 6.80. In order to determine the total extent of lipolysis, back-titration experiments at pH 11.50 were performed. Results suggest that there is a specific chain length range (C2–C8) for which pancreatic lipase shows higher activity. This specificity could result from a combination of physicochemical properties of TGs, 2-monoglycerides (2-MGs) and FAs, namely the droplet size of the TGs, the solubility of 2-MGs within mixed micelles, and the relative stability of the FAs as leaving groups in the hydrolysis reaction. During experimentation, it was evident that an optimisation of lipolysis conditions was needed for tighter control over pH levels so as to better mimic in vivo conditions. 1 M NaOH, 3.5 mL/min maximum dosing rate, and 3 μL/min minimum dosing rate were the optimised set of conditions that allowed better pH control, as well as the differentiation of the lipolysis of different lipid loads

Expression and functionality of P-glycoprotein in human bronchial epithelial cells in vitro

P-glycoprotein (P-gp) is expressed in normal tissues with barrier functions where it participates in cell defence mechanisms (Huls, M. et al. J Pharm Exp Ther 2009; 328:3-9). Its presence in the bronchial epithelium and role in the lung protection against inhaled toxicants has yet to be elucidated. The human bronchial epithelial cell line Calu-3 and normal human bronchial epithelial (NHBE) cells were cultured at an air-liquid interface on Transwell® inserts for 21 days. P-gp expression was evaluated by quantitative polymerase chain reaction and its functionality was assessed by permeability measurements using the established substrate 3H-digoxin either alone or in the presence of chemical or biomolecular inhibitors. P-gp was absent in NHBE cells and moderately expressed in Calu-3 cells. Net secretory transport of 3H-digoxin was observed in both models. This was reduced at 4°C and in the presence of the selective but non specific P-gp inhibitor PSC833 and the multidrug resistance protein (MRP) inhibitor MK571. The P-gp specific antibody inhibitor UIC2 and the metabolic inhibitors sodium azide and sodium dichloroacetate had no effect on 3H-digoxin transport in Calu-3 cells. The presence of active transport mechanisms in cultures of human bronchial epithelial cells was demonstrated, although they differed between the models tested. P-gp was not detected in NHBE cells, in line with observed low gene expression in human lung tissue (Bleasby, K. et al. Xenobiotica 2006; 36:963-988). The involvement of P-gp could not be confirmed and the transporter(s) responsible for 3H-digoxin asymmetric broncho-epithelial permeability remain(s) to be identified.Peer reviewe

Evaluation of layers of the rat airway epithelial cell line RL-65 for permeability screening of inhaled drug candidates.

A rat respiratory epithelial cell culture system for in vitro prediction of drug pulmonary absorption is currently lacking. Such a model may however enhance the understanding of interspecies differences in inhaled drug pharmacokinetics by filling the gap between human in vitro and rat in/ex vivo drug permeability screens. The rat airway epithelial cell line RL-65 was cultured on Transwell® inserts for up to 21days at an air-liquid (AL) interface and cell layers were evaluated for their suitability as a drug permeability measurement tool. These layers were found to be morphologically representative of the bronchial/bronchiolar epithelium when cultured for 8days in a defined serum-free medium. In addition, RL-65 layers developed epithelial barrier properties with a transepithelial electrical resistance (TEER) >300Ωcm(2) and apparent (14)C-mannitol permeability (P(app)) values between 0.5-3.0×10(-6)cm/s; i.e., in the same range as established in vitro human bronchial epithelial absorption models. Expression of P-glycoprotein was confirmed by gene analysis and immunohistochemistry. Nevertheless, no vectorial transport of the established substrates (3)H-digoxin and Rhodamine123 was observed across the layers. Although preliminary, this study shows RL-65 cell layers have the potential to become a useful in vitro screening tool in the pre-clinical development of inhaled drug candidates.Peer reviewe

Characterisation of ATP-binding cassette (ABC) transporters in the Calu-3 human bronchial epithelial cell culture model

Expression and functionality of ATP-binding cassette (ABC) transporters were assessed in Calu-3 cell monolayers cultured at an air-liquid interface. Transporter gene expression was in agreement with published data in human lungs with the exception of BSEP which was over-expressed in Calu-3 cells. Net secretory transport of the ABC substrate 3H-digoxin was reduced in presence of the inhibitors verapamil, PSC833 and MK571. However, the transporter(s) involved could not be identified.Peer reviewe

An Evaluation of the Pharmacodynamics, Safety, and Tolerability of the Potassium Binder RDX7675

Hyperkalemia is common in patients with heart failure or chronic kidney disease, particularly those taking renin‐angiotensin‐aldosterone system inhibitors, and can cause arrhythmias and sudden cardiac death. The most widely used treatment, sodium polystyrene sulfonate (SPS), limits gastrointestinal potassium absorption, but has poor palatability. RDX7675 (RDX227675) is the calcium salt of a reengineered polystyrene sulfonate‐based resin with improved palatability over SPS. The pharmacodynamic effects and safety of RDX7675 were assessed in a phase 1, single‐center, randomized, active‐controlled study. Healthy volunteers received nominal active doses of RDX7675 4.6 g twice a day (BID), 4.6 g 3 times a day (TID), 6.9 g BID, 13.7 g daily (QD), 9.2 g TID, or 13.7 g BID (n = 12 each), or equivalent doses of SPS (n = 3 each), for 4 days. RDX7675 dosing increased stool potassium excretion and decreased urinary potassium excretion from baseline. Stool potassium excretion increased by up to 1481 mg/day with RDX7675 (6.9 g BID), and urinary potassium excretion decreased by up to 939 mg/day (13.7 g BID). Similar levels of potassium excretion were observed using QD, BID, or TID dosing of a 13.7 g total daily RDX7675 dose. Few adverse events were reported. In conclusion, repeated oral dosing with RDX7675 over 4 days reduced potassium absorption in healthy volunteers; the results support QD dosing of RDX7675 in future clinical studies