Prediction of fraction metabolized via CYP3A in humans utilizing cryopreserved human hepatocytes from a set of 12 single donors.

1. It has previously been demonstrated that metabolism of drugs via a single enzymatic pathway, particularly CYP3A4, is associated with increased risk for drug-drug interactions (DDI). Quantitative experimental systems as well as integrated prediction models to assess such risk during the preclinical phase are highly warranted. 2. The present study was designed to systematically investigate the performance of human cryopreserved hepatocytes in suspension to predict fraction metabolized via CYP3A (fmCYP3A) by assessing the ketoconazole sensitive intrinsic clearance (CLint) for five prototypical CYP3A substrates with varying degree of CYP3A dependent CLint in twelve individual hepatocyte batches. 3. We demonstrate that in contrast to well predicted mean hepatic metabolic clearance (CLH) and mean fmCYP3A data, the variability in CYP3A contribution for compounds having multiple metabolic pathways cannot be predicted from inhibition experiments using ketoconazole as inhibitor. Instead, data in the present paper indicate that the variability is larger after inhibition of CYP3A for compounds having multiple metabolic pathways. 4. It is therefore recommended to estimate the average CLint and fmCYP3A for a given test compound in a series (n = 10) of individual human hepatocyte batches.

Understanding the critical disposition pathways of statins to assess drug-drug interaction risk during drug development: it's not just about OATP1B1.

The use of statins is widespread across disease areas because many patients have comorbidities. Given that these drugs have become common as comedications, it is essential to have an understanding of the potential risks of drug-drug interactions (DDIs) between statins and candidate drugs in development. Although the hepatic uptake transporter organic anion-transporting polypeptide 1B1 (OATP1B1) is known to play a substantial role in statin-related DDI risk, other transporters and metabolizing enzymes can also be involved. Consequently, a holistic approach to risk assessment is required, tailored to each statin. Using evidence from pharmacogenetics, DDIs, and literature on absorption, distribution, metabolism, and elimination (ADME) in humans, this review identifies pathways that contribute the most to, and are therefore the most critical to, the disposition of each statin. It also provides an understanding of the expected theoretical maximum increase in systemic exposure if the disposition of a statin is inhibited. Finally, on a statin-by-statin basis, we propose in vitro inhibition studies that should be routinely conducted during drug development so as to better assess DDI risk.

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 21 days 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 8 days in a defined serum-free medium. In addition, RL-65 layers developed epithelial barrier properties with a transepithelial electrical resistance (TEER) >300 Ω cm(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.

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.

Characterization of binding properties to human P-glycoprotein: development of a [3H]verapamil radioligand-binding assay.

Interaction with the exsorptive transporter P-glycoprotein (P-gp) is a possible source of peculiarities in drug pharmacokinetics, including dose-dependent absorption, drug-drug interactions, intestinal secretion, and limited permeability of the blood-brain barrier. Among the established in vitro methods of the analysis of drug interactions with P-gp, none directly quantifies the affinity of ligands with P-gp. Instead, they measure the result of a membrane permeation and a receptor-binding process; this may lead to difficulties in the interpretation of results. An assay for quantification of drug affinity to the transporter is presented on the basis of the radioligand-binding assay principle. This has the advantage of directly quantifying the interaction between drugs and P-gp. Because of the reversible and competitive interaction of numerous substrates with P-gp, a radioligand-binding assay was developed by taking [3H]verapamil and [3H]vinblastine as radioligands and the human intestinal Caco-2 cells, overexpressed with P-gp by culturing in the presence of vinblastine or transfecting with multidrug resistance gene MDR-1 as receptor preparation. The assay was performed in 96-well plates and has the potential to be used as a high-throughput method. A clear induction of the expression of P-gp was demonstrated in the Caco-2 cells grown in the presence of vinblastine, as well as in the transfected cells, although to a lesser extent. Both radioligands were shown to bind to P-gp. Verapamil was the radioligand of choice for further investigations due to its lower nonspecific binding to the transporter preparation. Kinetics as well as specificity of the binding of verapamil to the P-gp preparation were demonstrated. A two-affinity model was found to adequately describe the data derived from saturation as well as from competition experiments, in accordance with previous findings on two exsorption sites for P-gp. The binding properties of [3H]verapamil and [3H]vinblastine to a P-gp preparation derived from induced Caco-2 cells are described. The concentration-dependent displacement of the radioligand by nonlabeled substrates for P-gp should be a suitable principle for the determination of drug affinity to the respective binding sites at the human intestinal multidrug transporter P-gp.

P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [3H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.