EVALUATION OF INHIBITORY POTENCIES FOR COMPOUNDS INHIBITING P-GLYCOPROTEIN BUT WITHOUT MAXIMUM EFFECTS: F₂ VALUES

Abstract

ABSTRACT:In cell culture systems with aqueous buffers, concentration-response curves to lipophilic inhibitors are difficult to establish because plateau effects (I max ) are often not reached because of limited drug solubility. Consequently, the inhibitory potency of a compound will not be definable using IC 50 values (concentration exerting 50% of I max ). Since alternative potency measures f 2 values, the concentrations required to double baseline signals have been proposed. Using both methods, we reevaluated the concentration-response curves of calcein assays with 78 compounds in three different cell culture systems and found a close correlation between both methods (r s ‫؍‬ 0.93-0.99, p < 0.0028). These findings suggest that f 2 values are a valuable alternative to define rank orders of highly lipophilic inhibitors as a basis for the prediction of pharmacological interaction properties in clinical settings. Although it was only tested for inhibition of P-glycoprotein, it seems likely that this method may be transferred to other assays with other proteins.The assessment of the concentration-response relationship is a key element in the pharmacologic characterization of inhibitors of important targets like the ATP-binding cassette (ABC) transporter Pglycoprotein (P-gp; MDR1/ABCB1). The inhibitory potency of a compound is normally expressed as IC 50 (concentration leading to half-maximal inhibition) and is typically derived from the Hill equation or its derivatives,which describe concentration-response curves using four parameters: background (I min ), maximum effect (I max ), slope (s), and IC 50 , the concentration leading to 50% of I max . Obviously, the accurate determination of IC 50 values depends on the reliable description of I min , s, and I max , with the latter being the most challenging, since maximum effects can often not be reached because of limited solubility or cytotoxic effects. We have previously shown that the poor solubility is frequently neglected and that numerous papers report having tested inhibition at drug concentrations beyond the maximum solubility of the inhibitor in the respective buffers (Weiss et al., 2002).In this study we have evaluated an alternative method to assess the inhibitory potency of compounds interacting with P-gp, the substrates of which are typically highly lipophilic and typically cannot be tested up to maximal effects in vitro because of their low solubility in the aqueous solutions used in cell culture systems. P-gp inhibition was investigated with the well established and widely used calcein assay in three different cell systems expressing moderate to high amounts of this efflux transporter: primary porcine brain capillary endothelial cells (pBCECs), L-MDR1 cells with overexpression of human P-gp, and the murine leukemic cell line P388/dx overexpressing murine P-gp. Materials and MethodsMaterials. Culture media, fetal calf serum, medium supplements, antibiotics, and Hanks' balanced salt solution were purchased from Invitrogen (Karlsruhe, Germany), dimethyl sul...

Interaction of Thiazolidinediones (Glitazones) with the ATP-Binding Cassette Transporters P-Glycoprotein and Breast Cancer Resistance Protein

“…They were analyzed using GraphPad Prism version 5.0 and InStat version 3.05 (GraphPad Software, San Diego, Calif., USA). For calculation of the inhibitor effects in the calcein assay, the f 2 value (concentration needed to increase baseline fluorescence by a factor of 2 8 SD) was calculated as previously described and validated [30,31] because no compound reached plateau effects. The differences in P-gp mRNA expression following drug incubation compared with the respective vehicle controls were tested using ANOVA with Dunnett's post-hoc test.…”

HepaRG Cells as an in Vitro Model for Evaluation of Cytochrome P450 Induction in Humans

“…The F 2 value is the concentration leading to a 2-fold increase of the baseline levels. This approach was used by Weiss and Haefeli (2006) and found useful for evaluation of inhibition of P-glycoprotein.…”

Examination of the Ability of the Nasal Administration Route to Confer a Brain Exposure Advantage for Three Chemical Inhibitors of P-Glycoprotein

“…Model compounds for this study were selected on the basis of their status as P-gp inhibitors 15,16 and diversity of physicochemical properties (Tab. 1).…”

“…However, GF120918 brain exposure was similar for the two routes of administration, despite the difference in administered dose. For the antiarrhythmic agent quinidine, which has modest P-gp inhibitory properties, 15 nasal delivery did not improve either brain partitioning or brain exposure.…”

“…1). [8][9][10][11][12][13][14] These model compounds were selected for their status as inhibitors of P-glycoprotein (P-gp), 15,16 which is the major efflux transporter limiting drug uptake at the BBB. 17 Numerous chemical inhibitors of P-gp have been developed.…”