29 research outputs found
The Effects of Pregnenolone 16α-Carbonitrile Dosing on Digoxin Pharmacokinetics and Intestinal Absorption in the Rat
The effect of Pgp induction in rats by pregnenolone 16α-carbonitrile (PCN) (3 days, 35 mg/kg/d, p.o.) on digoxin pharmacokinetics and intestinal transport has been assessed. After intravenous or oral digoxin dosing the arterial and hepatic portal vein (oral) AUC(0-24h) were significantly reduced by PCN pre-treatment. Biliary digoxin clearance increased 2-fold following PCN treatment. PCN significantly increased net digoxin secretion (2.05- and 4.5-fold respectively) in ileum and colon but not in duodenum or jejunum. This increased secretion correlated with increased Pgp protein expression in ileum and colon. Both intestinal and biliary excretion therefore contribute to altered digoxin disposition following PCN
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
Resolving the DistributionâMetabolism Interplay of Eight OATP Substrates in the Standard Clearance Assay with Suspended Human Cryopreserved Hepatocytes
Uptake transporters may act to elevate
the intrahepatic exposure
of drugs, impacting the route and rate of elimination, as well as
the drugâdrug interaction potential. We have here extended
the assessment of metabolic drug stability in a standard human hepatocyte
incubation to allow for elucidation of the distributionâmetabolism
interplay established for substrates of drug transporters. Cellular
concentrationâtime profiles were obtained from incubations
of eight known OATP substrates at 1 ÎŒM, each for two different
10-donor batches of suspended cryopreserved human hepatocytes. The
kinetic data sets were analyzed using a mechanistic mathematical model
that allowed for separate estimation of active uptake, bidirectional
diffusion, metabolism and nonspecific extracellular and intracellular
binding. The range of intrinsic clearances attributed to active uptake,
diffusion and metabolism of the test set spanned more than 2 orders
of magnitude each, with median values of 18, 5.3, and 0.5 ÎŒL/min/10<sup>6</sup> cells, respectively. This is to be compared with the values
for the apparent clearance from the incubations, which only spanned
1 order of magnitude with a median of 2.6 ÎŒL/min/10<sup>6</sup> cells. The parameter estimates of the two pooled 10-donor hepatocyte
batches investigated displayed only small differences in contrast
to the variability associated with use of cells from individual donors
reported in the literature. The active contribution to the total cellular
uptake ranged from 55% (glyburide) to 96% (rosuvastatin), with an
unbound intra-to-extracellular concentration ratio at steady state
of 2.1 and 17, respectively. Principal component analysis showed that
the parameter estimates of the investigated compounds were largely
influenced by lipophilicity. Active cellular uptake in hepatocytes
was furthermore correlated to pure OATP1B1-mediated uptake as measured
in a transfected cell system. The presented approach enables the assessment
of the key pathways regulating hepatic disposition of transporter
and enzyme substrates from one single, reproducible and generally
accessible human <i>in vitro</i> system
In Vitro Intrinsic Permeability: A Transporter-Independent Measure of Cacoâ2 Cell Permeability in Drug Design and Development
In
vitro permeability data have a central place in absorption risk
assessments in drug discovery and development. For compounds where
active efflux impacts permeability in vitro, the inherent passive
membrane permeability (âintrinsic permeabilityâ) gives
a concentration-independent measure of the compoundâs permeability.
This work describes the validation of an in vitro intrinsic permeability
assay and application of the data in a predictive in silico model.
Apparent intrinsic permeability (P<sub>app</sub>) across Caco-2 cell
monolayers is determined in the presence of an optimized cocktail
of chemical inhibitors toward the three major efflux transporters
ABCB1, ABCC2, and ABCG2. The intrinsic P<sub>app</sub> value gives
an estimate of passive permeability, which is independent of transporter
expression levels and not limited by solubility or cell toxicity.
An in silico model has been established to predict the Caco-2 intrinsic
permeability and shown to consistently identify highly permeable compounds.
The new intrinsic permeability assay is useful for early absorption
estimates and suitable for absorption risk assessment in DMPK and
pharmaceutical development
Utility of in vitro systems and preclinical data for the prediction of human intestinal first-pass metabolism during drug discovery and preclinical development
A growing awareness of the risks associated with extensive intestinal metabolism has triggered an interest in developing robust methods for its quantitative assessment. This study explored the utility of intestinal S9 fractions, human liver microsomes, and recombinant cytochromes P450 to quantify CYP3A-mediated intestinal extraction in humans for a selection of marketed drugs that are predominantly metabolized by CYP3A4. A simple competing rates model is used to estimate the fraction of drug escaping gut wall metabolism (f g ) from in vitro intrinsic clearance in humans. The f g values extrapolated from the three in vitro systems used in this study, together with literature-derived f g from human intestinal microsomes, were validated against f g extracted from human in vivo pharmacokinetic (PK) profiles using a generic whole-body physiologically-based pharmacokinetic (PBPK) model. The utility of the rat as a model for human CYP3A-mediated intestinal meta bolism was also evaluated. Human f g from PBPK compares well with that from the grapefruit juice method, justifying its use for the evaluation of human in vitro systems. Predictive performance of all human in vitro systems was comparable [root mean square error (RMSE) = 0.22-0.27; n = 10]. Rat f g derived from in vivo PK profiles using PBPK has the lowest RMSE (0.19; n = 11) for the prediction of human f g for the selected compounds, most of which have a fraction absorbed close to 1. On the basis of these evaluations, the combined use of f g from human in vitro systems and rats is recommended for the estimation of CYP3A4-mediated intestinal metabolism in lead optimization and preclinical development phases
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 TranswellŸ 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 TranswellÂź 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) >300âŠcm(2) and apparent (14)C-mannitol permeability (P(app)) values between 0.5-3.0Ă10(-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