title: Evaluation of cocktail approach to standardise Caco-2 permeability experiments.
abstract: The purpose of this study was to investigate the suitability and reliability of n-in-one approach using FDA suggested compounds for standardising Caco-2 permeability experiments. Special attention was paid to the evaluation of rank order correlation and mechanistic insights of compound permeability. Transport studies with antipyrine, metoprolol, ketoprofen, verapamil, hydrochlorothiazide, ranitidine, mannitol and fluorescein were performed in 12- and 24-well formats, as single compounds and in cocktails under iso-pH 7.4 and pH-gradient (pH 5.5 vs. 7.4) conditions. Compounds were quantified using n-in-one LC/MS/MS analysis. The cocktail-dosing proved to be a feasible method to determine the permeability of the Caco-2 cell line and to introduce external standards for permeability tests. Even though sink conditions were lost in cocktail experiments for highly permeable compounds, the rank order of compound permeability and the classification to low and high permeability compounds remained unchanged between single and cocktail studies and permeability values of 12- and 24-well formats were directly comparable. Under pH-gradient conditions the margin between high and low permeability compounds was narrower due to the lower permeability (higher fraction of ionisation) of basic molecules. Of the compounds studied, antipyrine, metoprolol, hydrochlorothiazide and mannitol are suitable for evaluation and standardisation purposes of passive permeability, while fluorescein would function as paracellular marker under iso-pH 7.4. As efflux activity may vary between cell batches, verapamil is a useful marker for P-glycoprotein.
authors.affiliation: Drug Discovery and Development Technology Center (DDTC), Faculty of Pharmacy, University of Helsinki, Finland. maija.koljonen@helsinki.fi
authors.forename: Maija
authors.initials: M
authors.lastname: Koljonen
authors.forename: Kati S
authors.initials: KS
authors.lastname: Hakala
authors.forename: Tuula
authors.initials: T
authors.lastname: Ahtola-Sätilä
authors.forename: Leena
authors.initials: L
authors.lastname: Laitinen
authors.forename: Risto
authors.initials: R
authors.lastname: Kostiainen
authors.forename: Tapio
authors.initials: T
authors.lastname: Kotiaho
authors.forename: Ann Marie
authors.initials: AM
authors.lastname: Kaukonen
authors.forename: Jouni
authors.initials: J
authors.lastname: Hirvonen
mesh_terms: D001693:Biological Transport, Active; D018938:Caco-2 Cells; D018929:Cell Culture Techniques / Q000592:standards*; D002463:Cell Membrane Permeability; D002851:Chromatography, High Pressure Liquid; D004058:Diffusion; D004353:Drug Evaluation, Preclinical / Q000592:standards*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007413:Intestinal Mucosa / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D004364:Pharmaceutical Preparations