The Elementary Mass Action Rate Constants of P-gp Transport for a Confluent Monolayer of MDCKII-hMDR1 Cells

Abstract

The human multi-drug resistance membrane transporter, P-glycoprotein, or P-gp, has been extensively studied due to its importance to human health and disease. Thus far, the kinetic analysis of P-gp transport has been limited to steady-state Michaelis-Menten approaches or to compartmental models, neither of which can prove molecular mechanisms. Determination of the elementary kinetic rate constants of transport will be essential to understanding how P-gp works. The experimental system we use is a confluent monolayer of MDCKII-hMDR1 cells that overexpress P-gp. It is a physiologically relevant model system, and transport is measured without biochemical manipulations of P-gp. The Michaelis-Menten mass action reaction is used to model P-gp transport. Without imposing the steady-state assumptions, this reaction depends upon several parameters that must be simultaneously fitted. An exhaustive fitting of transport data to find all possible parameter vectors that best fit the data was accomplished with a reasonable computation time using a hierarchical algorithm. For three P-gp substrates (amprenavir, loperamide, and quinidine), we have successfully fitted the elementary rate constants, i.e., drug association to P-gp from the apical membrane inner monolayer, drug dissociation back into the apical membrane inner monolayer, and drug efflux from P-gp into the apical chamber, as well as the density of efflux active P-gp. All three drugs had overlapping ranges for the efflux active P-gp, which was a benchmark for the validity of the fitting process. One novel finding was that the association to P-gp appears to be rate-limited solely by drug lateral diffusion within the inner monolayer of the plasma membrane for all three drugs. This would be expected if P-gp structure were open to the lipids of the apical membrane inner monolayer, as has been suggested by recent structural studies. The fitted kinetic parameters show how P-gp efflux of a wide range of xenobiotics has been maximized.

Kinetic Identification of Membrane Transporters That Assist P-glycoprotein-Mediated Transport of Digoxin and Loperamide through a Confluent Monolayer of MDCKII-hMDR1 Cells

“…MATLAB fminsearch minimizes the coefficient of variation between the data and the simulated curves. Further details can be found in Tran et al (2005) and Bentz et al (2005).…”

“…Polarized confluent cell monolayers overexpressing P-gp have been used extensively as a model system to study P-gp transport mechanisms and to assess the risk of P-gp-mediated drug-drug interactions (Tang et al, 2002;Rautio et al, 2006;Bartholomé et al, 2007;Korjamo et al, 2007). Using the polarized Madin-Darby canine kidney II cell line that overexpresses human MDR1 (MDCKII-hMDR1) confluent cell monolayer, which overexpresses human P-gp in the apical plasma membrane and a detailed mass action analysis of P-gp efflux kinetics, we have been able to answer several fundamental questions about P-gp function Tran et al, 2005;Acharya et al, 2006). With P-gp as the only transporter in the kinetic model, we have found that amprenavir and quinidine were well fitted, whereas loperamide showed more efflux than could be explained by just P-gp (Tran Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.…”

“…This lipid composition is a rough mimic for the inner apical monolayer, as described in Tran et al (2005). e Steady-state passive permeability coefficients measured across the confluent cell monolayer in the presence of the P-gp inhibitor GF120918.…”

“…For many substrates, including those studied here, passive permeability is a significant fraction of total transport and is quantitatively analyzed separately using the P-gp inhibitor, GF120918 (Hyafil et al, 1993;Tran et al, 2005;Acharya et al, 2006). Our operational definition of passive permeability is ϩGF120918, which completely inhibits P-gp and some other transporters, both known and novel.…”

Intestinal transporters for endogenic and pharmaceutical organic anions: the challenges of deriving in-vitro kinetic parameters for the prediction of clinically relevant drug–drug interactions

“…[196] The ongoing transgression of Michaelis-Menten kinetics for determining kinetic constants for efflux transporters and the difficulties in estimating the enterocytic concentration has been recognized by several groups that have attempted to derive Ki and Km values from more or less complex three-compartment models and simulation models. [30,189,190,[196][197][198][199] However, a thorough validation of these models is still lacking and it is outside the scope of this review to further address these models. As emphasized by Tachibana et al [193] , there is a need of standardizing both the in-vitro methods and the method used for the Ki/IC50 calculations.…”