title: A P-gp vesicular transport inhibition assay - optimization and validation for drug-drug interaction testing.
abstract: Accurate determination of potential drug-drug interaction mediated by efflux transporters (tDDI) is crucial to assess the risk of pharmacokinetic interaction and toxicity of drugs. Passive permeability and uptake transporter mediated transport are important covariates of cell-based inhibition assays that need to be taken into consideration when performing kinetic analysis of data. Vesicular uptake inhibition has been considered by regulatory agencies as a viable alternative for testing tDDI potential of low passive permeability drugs in particular. Membranes prepared from a P-gp overexpressing human cell line has superior transport properties over membranes prepared from Sf9 cells and cholesterol enriched Sf9 membranes. P-gp expressed in this membrane effluxes N-methyl-quinidine (NMQ) with high affinity (K(m) is 3.65 μM) and a high rate (V(max) is 656 pmol/mg protein/min). Digoxin, vinblastine and paclitaxel, established P-gp substrates inhibited transport of NMQ with estimated K(i) values of 250, 0.1 and 0.6 μM, respectively. A panel of 11 drugs that have been listed by regulatory agencies as reference inhibitors were used to validate the assay to predict clinical inhibition potential. All the drugs that have been implicated in P-gp mediated DDI were found to be inhibitors in a relevant concentration range.
authors.affiliation: Solvo Biotechnology, Középfasor 52, 6726 Szeged, Hungary.
authors.forename: Krisztina
authors.initials: K
authors.lastname: Herédi-Szabó
authors.forename: Johan E
authors.initials: JE
authors.lastname: Palm
authors.forename: Tommy B
authors.initials: TB
authors.lastname: Andersson
authors.forename: Ákos
authors.initials: Á
authors.lastname: Pál
authors.forename: Dóra
authors.initials: D
authors.lastname: Méhn
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Fekete
authors.forename: Erzsébet
authors.initials: E
authors.lastname: Beéry
authors.forename: Katalin Tauberné
authors.initials: KT
authors.lastname: Jakab
authors.forename: Márton
authors.initials: M
authors.lastname: Jani
authors.forename: Peter
authors.initials: P
authors.lastname: Krajcsi
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001681:Biological Assay; D001692:Biological Transport; D002460:Cell Line; D004347:Drug Interactions; D006801:Humans; D007313:Insecta; D020014:K562 Cells; D004364:Pharmaceutical Preparations / Q000378:metabolism; D011804:Quinolines / Q000378:metabolism*; D015203:Reproducibility of Results; D022161:Transport Vesicles
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D004364:Pharmaceutical Preparations; D011804:Quinolines
keywords: ABCB1; DDI; Human cell membrane; N-methyl quinidine; P-glycoprotein; Vesicular uptake