Pgp ATPase assay

This study focused on potential effects on Pgp transporters. Additional transporters present in Caco-2 cell lines may lead to potential cross-reactivity, the respective assay hence relied on recombinant human Pgp transporters integrated in membrane fractions.

The surface active co-formulants and the solvent Solgad^® 150 ULN were tested at sub-cytotoxic concentrations for Pgp inhibition after stimulation with verapamil. Abamectin was tested for Pgp inhibition with verapamil, as well as for substrate properties without verapamil, since abamectin has been described as a mammalian Pgp substrate with inhibitory properties at higher concentrations (Lespine et al. 2007). Furthermore, fluroxypyr-meptyl was tested for Pgp substrate properties. Pgp ATPase activity modulation was determined using the Pgp-Glo assay system (Promega, Madison, WI, USA) following the manufacturer's user protocol. Briefly, 25 µg of recombinant human Pgp membrane in Pgp-Glo assay buffer was incubated with different concentrations of each active substance or co-formulant in combination with 200 µM verapamil at 37 °C for 5 min. Furthermore, Pgp membrane was incubated with 100 µM Na₃VO₄ (selective inhibitor), 200 µM verapamil (substrate) or Pgp-Glo assay buffer (negative control). 5 mM MgATP was added to each well and 96-well plates were incubated at 37 °C for 40 min to initiate the reaction. Luminescence of the remaining, unmetabolised ATP was initiated by adding 50 µl of 25 mM ATP detection reagent. After briefly shaking on a plate shaker, 96-well plates were incubated at room temperature for 20 min. The luciferase generated luminescence signal was measured on an Infinite M200 PRO plate reader (Tecan, Maennedorf, Swiss). All measurements were corrected by subtraction of Na₃VO₄-treated signals (non Pgp ATPase activity) and represented as changes in luminescence signals (ΔRLU). Basal Pgp ATPase activity is represented by the difference in luminescence signals between Na₃VO₄-treated samples and untreated samples. Changes in luminescence (ΔRLU) caused by the co-formulants and abamectin incubated with verapamil were compared to ΔRLU caused by verapamil. ΔRLU values of the active ingredients (without verapamil) were compared to ΔRLU values of untreated samples (basal ΔRLU). Each condition was measured in four replicates (n = 4).