If the K_I Is Defined by the Free Energy of Binding to P-Glycoprotein, Which Kinetic Parameters Define the IC₅₀ for the Madin-Darby Canine Kidney II Cell Line Overexpressing Human Multidrug Resistance 1 Confluent Cell Monolayer?

Abstract

ABSTRACT:From previous fits of drug transport kinetics across confluent Madin-Darby canine kidney II cell line overexpressing human multidrug resistance 1 cell monolayers, we found that a drug's binding constant to P-glycoprotein (P-gp) was significantly smaller than its IC 50 when that drug was used as an inhibitor against another P-gp substrate. We tested several IC 50 candidate functions, including the standard function, the Kalvass-Pollack function, and the efflux ratio, to determine whether any of them yielded an IC 50 ‫؍‬ K I , as would be expected for water-soluble enzymes. For the confluent cell monolayer, the IC 50 /K I ratio is greater than 1 for all candidate functions tested. From the mass action kinetic model, we have derived a simple approximate equation that shows how the IC 50 /K I ratio depends on the elementary rate constants from our mass action model. Thus, the IC 50 will differ between cell lines and tissues, for the same probe substrate and inhibitor, if there are different membrane concentrations of P-gp, or the probe substrate's elementary rate constants, partition coefficient, binding constant to P-gp, passive permeability, and ability to access the other transporters (if any) in the two cell lines. The mass action model and the approximate equation for the IC 50 /K I ratio derived here can be used to estimate the elementary rate constants needed to extrapolate in vitro drug-drug interactions for compounds to the in vivo environment.

Variability in P-Glycoprotein Inhibitory Potency (IC50) Using Various in Vitro Experimental Systems: Implications for Universal Digoxin Drug-Drug Interaction Risk Assessment Decision Criteria

“…In the absence of inhibitor, cell line 2 will have a smaller intracellular probe substrate concentration than cell line 1, so the fraction of P-gp with substrate bound to it in the absence of inhibitor will be smaller in cell line 2. This means that more inhibitor will have to be added to cell line 2 to reduce the fraction of P-gp with substrate bound to it by 50% (Lumen et al, 2010). Thus, the IC 50 value for cell line 2 will be higher than the IC 50 value for cell line 1.…”

“…B1 (green squares) and D (black circles), with the exception of Inhibitor 15 (verapamil). It has been noted previously for MDCKII-MDR1 cells as well as LLC-PK 1 -MDR1 cells (Lumen et al, 2010, Sugimoto et al, 2011) that an IC 50 based on transport activity expressed as an efflux ratio (eq. A1 or A2) is typically lower than one based on B.A transport (B1).…”

“…The IC 50 value for P-gp in confluent monolayers is a complicated function of substrate and inhibitor efflux and influx kinetics (Lumen et al, 2010). For example, consider two cell lines identical in all respects except that the P-gp surface density in cell line 2 is greater than that of cell line 1.…”

Fitting the Elementary Rate Constants of the P-gp Transporter Network in the hMDR1-MDCK Confluent Cell Monolayer Using a Particle Swarm Algorithm

“…For the confluent cell monolayer, when the dissociation constant of the inhibitor to P-gp is denoted K I , we have shown that the ratio of the IC 50 / K I increases with increased P-gp surface density and probe-substrate elementary efflux rate constants and decreases with the contributions of other probe-substrate transporters [28]. The simple IC 50 analysis is very different with confluent cell monolayers, or tissue, than it is with water soluble enzymes, upon which the standard IC 50 equation were tested [28].…”

P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions

“…However, a second study measuring the cellular efflux of the P-gp substrate doxorubicin in MDR1 expressing B16/10 murine melanoma cells reported that propiconazole inhibited P-gp transport significantly less than verapamil (Bain and LeBlanc,1996). Such differences are not unexpected as P-gp and other endogenous transporters may be expressed at varying levels between cell line and passage number (Lumen et al, 2010). In addition, differing physicochemical properties of probe substrates can influence the potency of a xenobiotic to inhibit P-gp transport in a given assay (Zastre et al, 2008).…”